Graham JB , Sunryd JC , Mathavan K , Weir E , Larsen ISB , Halim A , Clausen H , Cousin H , Hebert DN .

R01 GM086874 NIGMS NIH HHS , T32 GM008515 NIGMS NIH HHS

	FIGURE 1: TMTC3 and TMTC4 are ER resident proteins. (A) The organization of TMTC3 and TMTC4 with signal sequences (black), hydrophobic domains (teal), and TPR domains (orange) as designated. The start and end positions of each domain are as follows: TMTC3 hydrophobic domains (amino acids 9–30, 94–115, 140–161, 169–187, 192–214, 235–257, 321–340, 347–367, 371–393, and 402–422), TMTC3 TPR motifs (amino acids 412–445, 446–479, 480–513, 529–562, 563–596, 597–630, 632–665, 669–702, 703–736, 738–771, and 772–805), TMTC4 hydrophobic domains (amino acids 20–38, 109–130, 142–161, 170–192, 203–221, 225–245, 269–291, 320–339, 353–374, 381–403, 412–433, and 440–460), and TMTC4 TPR motifs (amino acids 448–481, 482–515, 516–549, 550–583, 584–617, 618–651, 652–685, and 686–719). Predicted endogenous N-linked glycosylation sites are indicated by small black, branched structures. (B) HEK293T cells were transfected with S-tagged TMTC3 or TMTC4 as indicated and were affinity purified from the cell lysate and media using S-protein agarose. Samples were then subjected to a glycosylation assay with either EndoH (lanes 2, 5, 8, 11, 14, and 17) or PNGaseF (lanes 3, 6, 9, 12, 15, and 18) digestion as indicated. Reducing sample buffer was added and the samples were analyzed by a 6% (TMTC3 S-tag) and 8% (TMTC4 S-tag) SDS–PAGE. (C) Cellular localization of TMTC3 and TMTC4 was investigated by confocal microscopy. COS7 cells were transfected with TMTC3 or TMTC4 cDNA. Fixed cells were stained with S-tag, ERp57 (ER) or GM130 (Golgi) antisera. Nuclei were visualized by 4′,6-diamidino-2-phenylindole staining (blue). Scale bars correspond to 10 µm.
	FIGURE 2: TMTC3 and TMTC4 are transmembrane proteins with their TPR domains facing the ER lumen. (A) HEK293T cells were transfected with S-tagged TMTC3 or TMTC4 as indicated and radiolabeled for 1 h with [35S]-Cys/Met. Cells were homogenized and fractionated prior to alkaline extraction. The fractions collected were whole cell lysate (WCL), nucleus (N), cytosol (C), total membrane (TM), as well as supernatant (S), and pellet (P) fractions on alkaline extraction of the TM. All samples were subjected to affinity purification with S-tag agarose or calnexin (CNX) and CRT immunoprecipitation where indicated. Samples were analyzed via SDS–PAGE and detected via autoradiography. (B) TMTC3 S-tag and TMTC4 S-tag were expressed in HEK293T cells. Cells were homogenized and microsomes were isolated by ultracentrifugation then resuspended in homogenization buffer. Aliquots of the ER microsomes were incubated for 15 min at 27°C with or without Triton X-100 and trypsin where indicated. Samples were separated on 9% SDS–PAGE and immunoblotted for the S-tag epitope. (C) Putative trypsin protease cleavage areas (dashed line) on TMTC3 and TMTC4 based on exposed potential cleavage residues and size of cleaved bands in B. Orange hexagons represent ER lumen–facing TPR motifs.
	FIGURE 3: TMTC3 rescues O-mannosylation of E-cadherin in HEK293SC/TMTC1,2,3,4 cells. (A) HEK293T cells expressing TMTC1, -2, -3, or -4 were harvested in isotonic buffer and homogenized. A portion of the cell homogenate was subjected to ultracentrifugation and resuspended in reducing sample buffer. This was considered the total membrane fraction (lanes 1–5). An excess of MNT lysis buffer was added to an equal amount of cell homogenate and subjected to S-protein agarose affinity purification (lanes 6–10). Proteins were detected by immunoblotting with appropriate antisera directed against the S-tag epitope and SERCA2B. (B) S-tagged TMTC1, -2, -3, or -4 cDNA was transfected into indicated cell lines. Cell lysates were collected and split, one half subjected to treatment with endoglycosidase PNGaseF (lanes 2, 4, 6, 8, and 10), prior to pulling down glycosylated proteins with Con A. Samples were then analyzed by 6% SDS–PAGE and immunoblotted with E-cadherin antisera to assess levels of glycosylated E-cadherin. (C) Quantification of relative density of O-glycosylated E-cadherin from HEK293SC/TMTC1,2,3,4 cells transfected with TMTC1, -2, -3, and -4 cDNA, respectively. Statistical significance between nontransfected cells and TMTC1, -2, -3, or -4 transfected cells was calculated using one-way ANOVA. Measurements designated ** have a P value of <0.01. Error bars represent SD. (D) HEK293SC/TMTC1,2,3,4 cells were transfected with S-tagged TMTC3 cDNA. Cell lysates were collected and subjected to a glycosylation assay with either PNGaseF (lane 2) or combined PNGaseF and α1-2,3,6 mannosidase treatment (lane 3) prior to affinity purification of glycosylated proteins by Con A. Samples were then analyzed via 9% SDS–PAGE and immunoblotted with E-cadherin antisera. Error bars represent SD.
	FIGURE 4: TMTC3 enhances E-cadherin–mediated cellular adherence. (A) Cellular localization of cadherins was investigated by confocal microscopy. HEK293SC, HEK293SC/POMT1,2, and HEK293SC/TMTC1,2,3,4 cells were fixed and stained with pan-cadherin antisera. Scale bars correspond to 50 µm. (B) Adherence was assessed in previously described cell lines. Cells were resuspended in growth media, and 1 × 106 cells were subsequently plated in a 24-well plate. Cells were allowed to adhere for 30 min. Unadhered cells were collected and adhered cells were trypsinized and collected. The data describe the percentage of floating (unadhered) cells over the total (unadhered + adhered) after counting both fractions. Statistical significance between cell types was calculated using one-way ANOVA. Measurements designated ** have a P value of <0.01. Error bars represent the SD. (C) TMTC1, -2, -3, or -4 cDNA was transfected into HEK293SC/TMTC1,2,3,4 cells and adherence was assessed as described in B. The increase or decrease of adherence of cells transfected with TMTC1, -2, -3, or -4 was established by subtracting the percentage of mock transfected adhered cells (red dashed line) from the percentage of adhered, transfected cells (adhered/[unadhered + adhered]). Error bars represent the SD. Statistical significance between cell treatments was calculated using one-way ANOVA. Measurements designated *, **, or *** have a P value of <0.05, <0.01 or <0.001, respectively. (D) Cellular adherence to immobilized E-cadherin was assessed via immunoassay. HEK293SC, HEK293SC/POMT1,2, and HEK293SC/TMTC1,2,3,4 cells and HEK293SC/TMTC1,2,3,4 cells transfected with TMTC3 were resuspended and 60,000 cells were placed on immobilized E-cadherin (50 µg/ml). Cells were allowed to attach for 1 h prior to fixing with PFA, permeabilization with Triton X-100, and staining with Hoescht. Hoescht nuclear staining and DIC images (pictured) were acquired at 40× magnification. Scale bars correspond to 50 µm. (E) The number of cells adhered to E-cadherin in previously described cell lines was estimated by averaging five randomized images capturing nuclei intensity at 405 nm within each well from three independent experiments. Error bars represent the SD.
	FIGURE 5: TMTC3 WT and disease variant stability. (A) HEK293T cells were transfected with S-tagged TMTC3 WT or disease variant cDNA as indicated. Cells were treated with 100 μg/ml cycloheximide for the indicated time prior to collection and lysed in MNT, and samples were subjected to affinity purification with S-protein agarose. Reducing sample buffer was added and samples were analyzed via 9% SDS–PAGE and immunoblotted for the S-tag epitope. Cell lysates were also subjected to precipitation with 10% TCA, analyzed via 9% SDS–PAGE, and immunoblotted with KDEL antisera to determine stability of GRP94 and BiP (WCL). (B) Time points were collected at 0, 2, 4, 8, 16, and 24 h after cycloheximide treatment. The amount of TMTC3 protein remaining at 2, 4, 8, 16, and 24 h was quantified and normalized to the starting material (0 h) and averaged from three independent experiments. Error bars represent SEM. (C) HEK293T cells were transfected with S-tagged TMTC3 WT or the TMTC3 R488Efs disease variant as indicated. Cells transfected with the TMTC3 R488Efs variant were treated with 20 μM MG132 for 12 h prior to collection and lysis in MNT (lane 4). Samples were subjected to affinity purification with S-protein agarose and analyzed via SDS–PAGE prior to immunoblotting for the S-tag epitope.
	FIGURE 6: Knockdown of Tmtc3 in developing X. laevis emrbyos affects gastrulation. (A) Embryos were injected with 10 ng of morpholinos against Tmtc1, Tmtc2, Tmtc3, and Tmtc4 at the one-cell stage. The embryos were scored for gastrulation when control embryos reached stages 11–12. Embryos were also injected with 10 ng of Tmtc3 morpholino and subsequently injected with S-tagged huTMTC3WT, huTMTC3DD>AA huTMTC3EE>AA, huTMTC3H67D, huTMTC3R71H, and huTMTC3G384E at the one-cell stage. The embryos were scored as described above. (B) The average of three or more independent experiments is plotted on the graph. Statistical significance between injection conditions was calculated using one-way ANOVA. Measurements designated *** have a P value of < 0.001. The error bars represent the SD to the mean. The number of embryos analyzed are as follows: noninjected n = 75, TMTC1MO n = 43, TMTC2MO n = 45, TMTC3MO n = 43, TMTC4MO n = 40, TMTC3MO + huTMTC3 n = 35, TMTC3MO + huTMTC3DD>AAn = 33, TMTC3MO + huTMTC3EE>AAn = 30, TMTC3MO + huTMTC3H67Dn = 28, TMTC3 MO + huTMTC3R71Hn = 22, and huTMTC3G384En = 33.
	Supplemental Figure 1. TMTCs and POMTs share similar functional domain architecture. (A) The organization of E-cadherin with signal sequence (black), extracellular cadherin domains (pink) and transmembrane domain (teal) as designated. N-linked glycosylation sites are indicated by small black, branched structures and O-mannosylation sites by green circles. (B) The organization of yeast Pmt1 and predicted organization of human POMT1 and 2 with signal sequences (black), hydrophobic domains (teal) and MIR domains (blue) as designated. Predicted endogenous N-linked glycosylation sites are indicated by small black, branched structures and putative diacidic motif active sites are indicated in dark blue (red for active site that has been identified in yPmts).(C) Predicted organization of human TMTC3 with signal sequence (black), hydrophobic domains (teal), TPR domains (orange) and predicted active sites (diacidic DE and EE residues in dark blue) as designated.
	Supplemental Figure 2. Basal transcript abundance and fold induction of TMTC proteins by ER stress. (A) RNA from HEK293A cells, grown under normal conditions, was harvested. RNA was reverse transcribed to cDNA followed by qRT-PCR with appropriate primers. Basal mRNA abundance was assessed using β-actin as a reference. Error bars represent standard error from at least three independent experiments. (B) HEK293A cells were treated with regular growth media or with 2 mM DTT for 2 hr, 1 μg/mL tunicamycin, 3 μM thapsigargin, 2.5 μg/mL brefeldin A or 2.5 μM MG132 for 24 hr prior to RNA purification. RNA was reverse transcribed to cDNA followed by qRT-PCR with appropriate primers, and changes in gene expression were calculated using β-actin as a reference. Statistical significance between treatment groups was determined using an unpaired T test. * and ** indicates a P-value of less than 0.05 and 0.01, respectively. Error bars represent standard deviation from at least three independent experiments.
	Supplemental Figure 3. Deletion of TMTCs and E-cadherin. (A) S-tagged TMTC1, 2, 3 or 4 cDNA was transfected into HEK293SC/TMTC1,2,3,4 cells. Cell lysates were collected and one third was subjected to affinity purification with S-protein agarose. Samples were then analyzed by 6% SDS-PAGE and immunoblotted for the S-tag epitope to assess levels of TMTC1, 2, 3 and 4, respectively. (B) HEK293SC/TMTC1,2,3,4 cells were transfected with S-tagged TMTC3 WT or putative active site mutant (DD>AA or EE>AA) cDNA. Lysates were collected and split, one third subjected to treatment with endoglycosidase PNGaseF (lanes 2, 4, 6, 8 and 10), prior to pulling down glycosylated proteins with concanavalin A (Con A). One third was subjected to affinity purification with S-protein agarose. Samples were then analyzed by 6% SDS-PAGE and immunoblotted with E-cadherin antisera (top blot) or S-tag antisera to assess levels of TMTC3 (lower blots). Lower panel represents the upper panel S-tag blot at a higher exposure to see the diminished expression of the DD>AA mutant (lane 3) (C) HEK293T cells were transfected with S-tagged TMTC3 WT or putative active site mutant cDNA as indicated. Cells were treated with 100 μg/mL cycloheximide for the indicated time prior to collection in lysis buffer. Samples were subjected to affinity purification with S-protein agarose and analyzed via 9% SDS-PAGE and immunoblotted for the S-tag epitope. Time points were collected at 0, 2, 4, 8, 16 and 24 hr after cycloheximide treatment. The amount of TMTC3 protein remaining at 2, 4, 8, 16 and 24 hr was quantified and normalized to the starting material (0 h) and averaged from three independent experiments. Error bars represent standard error of the mean (upper panel).
	Supplemental Figure 4. Deletion of TMTCs and E-cadherin. (A) Expression and stability of E-cadherin was assessed in O-mannosyltransferase knockout cells to determine effects of O-glycosylation. HEK293SC, HEK293SC/POMT1,2 and HEK293SC/TMTC1,2,3,4 cells were lysed and total protein amount was assessed via Bradford assay. Equal amounts of protein were subjected to pull-down via Con A prior to samples being analyzed by 9% SDS-PAGE and immunoblotted with E-cadherin antisera to monitor levels of E-cadherin. (B) Previously described cell lines were treated with 100 μg/mL cycloheximide for indicated time (0, 2, 4, 8, 16 and 24 hr) prior to lysis, total protein quantification and subsequent affinity purification with Con A. Samples were then analyzed by 9% SDS-PAGE (left panel) and the amount of E-cadherin remaining at 2, 4, 8, 16 and 24 hr was quantified and normalized to the starting material (0 h) (right panel). Error bars represent standard error of the mean.
	Supplemental Figure 5. TMTC3 disease variant predictive outcomes and carbohydrate analysis. (A) The organization of TMTC3 WT and disease variants signal sequence (black), hydrophobic domains (teal), TPR domains (orange). N-linked glycosylation sites are indicated by small black, branched structures. Mutations are indicated in blue. (B) HEK293T cells were transfected with S-tagged TMTC3 WT and disease variants cDNA as indicated and were affinity purified from the media and the lysed cells using S-protein agarose. Samples were then subjected to a glycosylation assay with either EndoH (lanes 2, 5, 8 and 11) or PNGaseF (lanes 3, 6, 9 and 12) digestion as indicated. Reducing sample buffer was added, and the samples were analyzed by a 6% SDS-PAGE.
	Supplemental Figure 6. Cellular localization of TMTC3 disease variants. Cellular localization of TMTC3 disease variants was investigated by confocal microscopy. Cos7 cells were transfected with TMTC3 disease variant cDNA. Fixed cells were stained with S-tag, ERp57 (ER) or GM130 (Golgi) antisera. Nuclei were visualized by DAPI staining (blue).
	Supplemental Figure 7. TMTC conservation in commonly studied species and RNA expression in Xenopus laevis. (A) Sequence identity (%) of the TMTC proteins in commonly studied species was assessed by comparing amino acid sequences of each species listed to the human sequence. (B) RNA expression of Tmtc1-4 during Xenopus laevis development up to stage 40. These expression profiles were based on data collected in the study by Session et. al in Nature 2016. (C) Embryos were injected with Tmtc3 morpholino at the one cell stage approximately 45 min after fertilization. Selected embryos were subsequently injected with human TMTC3 RNA (lanes 3, 4, 5, 8, 9, 10 and 11). Non-injected and indicated injected embryos were collected at stage 12 and resuspended in Modified Barth’s Saline (MBS) buffer with 1% triton X-100. Total protein was precipitated with 10% trichloroacidic acid, resuspended in reducing sample buffer, analyzed by a 9% SDS-PAGE and immunobloted for the S-tag epitope.
	FIGURE 1:. TMTC3 and TMTC4 are ER resident proteins. (A) The organization of TMTC3 and TMTC4 with signal sequences (black), hydrophobic domains (teal), and TPR domains (orange) as designated. The start and end positions of each domain are as follows: TMTC3 hydrophobic domains (amino acids 9–30, 94–115, 140–161, 169–187, 192–214, 235–257, 321–340, 347–367, 371–393, and 402–422), TMTC3 TPR motifs (amino acids 412–445, 446–479, 480–513, 529–562, 563–596, 597–630, 632–665, 669–702, 703–736, 738–771, and 772–805), TMTC4 hydrophobic domains (amino acids 20–38, 109–130, 142–161, 170–192, 203–221, 225–245, 269–291, 320–339, 353–374, 381–403, 412–433, and 440–460), and TMTC4 TPR motifs (amino acids 448–481, 482–515, 516–549, 550–583, 584–617, 618–651, 652–685, and 686–719). Predicted endogenous N-linked glycosylation sites are indicated by small black, branched structures. (B) HEK293T cells were transfected with S-tagged TMTC3 or TMTC4 as indicated and were affinity purified from the cell lysate and media using S-protein agarose. Samples were then subjected to a glycosylation assay with either EndoH (lanes 2, 5, 8, 11, 14, and 17) or PNGaseF (lanes 3, 6, 9, 12, 15, and 18) digestion as indicated. Reducing sample buffer was added and the samples were analyzed by a 6% (TMTC3 S-tag) and 8% (TMTC4 S-tag) SDS–PAGE. (C) Cellular localization of TMTC3 and TMTC4 was investigated by confocal microscopy. COS7 cells were transfected with TMTC3 or TMTC4 cDNA. Fixed cells were stained with S-tag, ERp57 (ER) or GM130 (Golgi) antisera. Nuclei were visualized by 4′,6-diamidino-2-phenylindole staining (blue). Scale bars correspond to 10 µm.
	FIGURE 2:. TMTC3 and TMTC4 are transmembrane proteins with their TPR domains facing the ER lumen. (A) HEK293T cells were transfected with S-tagged TMTC3 or TMTC4 as indicated and radiolabeled for 1 h with [35S]-Cys/Met. Cells were homogenized and fractionated prior to alkaline extraction. The fractions collected were whole cell lysate (WCL), nucleus (N), cytosol (C), total membrane (TM), as well as supernatant (S), and pellet (P) fractions on alkaline extraction of the TM. All samples were subjected to affinity purification with S-tag agarose or calnexin (CNX) and CRT immunoprecipitation where indicated. Samples were analyzed via SDS–PAGE and detected via autoradiography. (B) TMTC3 S-tag and TMTC4 S-tag were expressed in HEK293T cells. Cells were homogenized and microsomes were isolated by ultracentrifugation then resuspended in homogenization buffer. Aliquots of the ER microsomes were incubated for 15 min at 27°C with or without Triton X-100 and trypsin where indicated. Samples were separated on 9% SDS–PAGE and immunoblotted for the S-tag epitope. (C) Putative trypsin protease cleavage areas (dashed line) on TMTC3 and TMTC4 based on exposed potential cleavage residues and size of cleaved bands in B. Orange hexagons represent ER lumen–facing TPR motifs.
	FIGURE 3:. TMTC3 rescues O-mannosylation of E-cadherin in HEK293SC/TMTC1,2,3,4 cells. (A) HEK293T cells expressing TMTC1, -2, -3, or -4 were harvested in isotonic buffer and homogenized. A portion of the cell homogenate was subjected to ultracentrifugation and resuspended in reducing sample buffer. This was considered the total membrane fraction (lanes 1–5). An excess of MNT lysis buffer was added to an equal amount of cell homogenate and subjected to S-protein agarose affinity purification (lanes 6–10). Proteins were detected by immunoblotting with appropriate antisera directed against the S-tag epitope and SERCA2B. (B) S-tagged TMTC1, -2, -3, or -4 cDNA was transfected into indicated cell lines. Cell lysates were collected and split, one half subjected to treatment with endoglycosidase PNGaseF (lanes 2, 4, 6, 8, and 10), prior to pulling down glycosylated proteins with Con A. Samples were then analyzed by 6% SDS–PAGE and immunoblotted with E-cadherin antisera to assess levels of glycosylated E-cadherin. (C) Quantification of relative density of O-glycosylated E-cadherin from HEK293SC/TMTC1,2,3,4 cells transfected with TMTC1, -2, -3, and -4 cDNA, respectively. Statistical significance between nontransfected cells and TMTC1, -2, -3, or -4 transfected cells was calculated using one-way ANOVA. Measurements designated ** have a P value of <0.01. Error bars represent SD. (D) HEK293SC/TMTC1,2,3,4 cells were transfected with S-tagged TMTC3 cDNA. Cell lysates were collected and subjected to a glycosylation assay with either PNGaseF (lane 2) or combined PNGaseF and α1-2,3,6 mannosidase treatment (lane 3) prior to affinity purification of glycosylated proteins by Con A. Samples were then analyzed via 9% SDS–PAGE and immunoblotted with E-cadherin antisera. Error bars represent SD.
	FIGURE 4:. TMTC3 enhances E-cadherin–mediated cellular adherence. (A) Cellular localization of cadherins was investigated by confocal microscopy. HEK293SC, HEK293SC/POMT1,2, and HEK293SC/TMTC1,2,3,4 cells were fixed and stained with pan-cadherin antisera. Scale bars correspond to 50 µm. (B) Adherence was assessed in previously described cell lines. Cells were resuspended in growth media, and 1 × 106 cells were subsequently plated in a 24-well plate. Cells were allowed to adhere for 30 min. Unadhered cells were collected and adhered cells were trypsinized and collected. The data describe the percentage of floating (unadhered) cells over the total (unadhered + adhered) after counting both fractions. Statistical significance between cell types was calculated using one-way ANOVA. Measurements designated ** have a P value of <0.01. Error bars represent the SD. (C) TMTC1, -2, -3, or -4 cDNA was transfected into HEK293SC/TMTC1,2,3,4 cells and adherence was assessed as described in B. The increase or decrease of adherence of cells transfected with TMTC1, -2, -3, or -4 was established by subtracting the percentage of mock transfected adhered cells (red dashed line) from the percentage of adhered, transfected cells (adhered/[unadhered + adhered]). Error bars represent the SD. Statistical significance between cell treatments was calculated using one-way ANOVA. Measurements designated *, **, or *** have a P value of <0.05, <0.01 or <0.001, respectively. (D) Cellular adherence to immobilized E-cadherin was assessed via immunoassay. HEK293SC, HEK293SC/POMT1,2, and HEK293SC/TMTC1,2,3,4 cells and HEK293SC/TMTC1,2,3,4 cells transfected with TMTC3 were resuspended and 60,000 cells were placed on immobilized E-cadherin (50 µg/ml). Cells were allowed to attach for 1 h prior to fixing with PFA, permeabilization with Triton X-100, and staining with Hoescht. Hoescht nuclear staining and DIC images (pictured) were acquired at 40× magnification. Scale bars correspond to 50 µm. (E) The number of cells adhered to E-cadherin in previously described cell lines was estimated by averaging five randomized images capturing nuclei intensity at 405 nm within each well from three independent experiments. Error bars represent the SD.
	FIGURE 5:. TMTC3 WT and disease variant stability. (A) HEK293T cells were transfected with S-tagged TMTC3 WT or disease variant cDNA as indicated. Cells were treated with 100 μg/ml cycloheximide for the indicated time prior to collection and lysed in MNT, and samples were subjected to affinity purification with S-protein agarose. Reducing sample buffer was added and samples were analyzed via 9% SDS–PAGE and immunoblotted for the S-tag epitope. Cell lysates were also subjected to precipitation with 10% TCA, analyzed via 9% SDS–PAGE, and immunoblotted with KDEL antisera to determine stability of GRP94 and BiP (WCL). (B) Time points were collected at 0, 2, 4, 8, 16, and 24 h after cycloheximide treatment. The amount of TMTC3 protein remaining at 2, 4, 8, 16, and 24 h was quantified and normalized to the starting material (0 h) and averaged from three independent experiments. Error bars represent SEM. (C) HEK293T cells were transfected with S-tagged TMTC3 WT or the TMTC3 R488Efs disease variant as indicated. Cells transfected with the TMTC3 R488Efs variant were treated with 20 μM MG132 for 12 h prior to collection and lysis in MNT (lane 4). Samples were subjected to affinity purification with S-protein agarose and analyzed via SDS–PAGE prior to immunoblotting for the S-tag epitope.
	FIGURE 6:. Knockdown of Tmtc3 in developing X. laevis emrbyos affects gastrulation. (A) Embryos were injected with 10 ng of morpholinos against Tmtc1, Tmtc2, Tmtc3, and Tmtc4 at the one-cell stage. The embryos were scored for gastrulation when control embryos reached stages 11–12. Embryos were also injected with 10 ng of Tmtc3 morpholino and subsequently injected with S-tagged huTMTC3WT, huTMTC3DD>AA huTMTC3EE>AA, huTMTC3H67D, huTMTC3R71H, and huTMTC3G384E at the one-cell stage. The embryos were scored as described above. (B) The average of three or more independent experiments is plotted on the graph. Statistical significance between injection conditions was calculated using one-way ANOVA. Measurements designated *** have a P value of < 0.001. The error bars represent the SD to the mean. The number of embryos analyzed are as follows: noninjected n = 75, TMTC1MO n = 43, TMTC2MO n = 45, TMTC3MO n = 43, TMTC4MO n = 40, TMTC3MO + huTMTC3 n = 35, TMTC3MO + huTMTC3DD>AA n = 33, TMTC3MO + huTMTC3EE>AA n = 30, TMTC3MO + huTMTC3H67D n = 28, TMTC3 MO + huTMTC3R71H n = 22, and huTMTC3G384E n = 33.

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