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	FIGURE 1. KV10.1 protein detection in melanoma cell lines. A, astemizole (red symbols) reduces proliferation (measured as percent confluence) in melanoma cell lines as compared with norastemizole (blue symbols) and vehicle control (black symbols). Both drugs were used at 500 nm. In IPC298 cells the proliferation was strongly reduced by astemizole treatment, whereas in IGR39 cells there was no effect. B, the cell extracts were precipitated using a mixture of two anti-KV10.1 monoclonal antibodies (mAb33 against the C terminus and mAb62 against the pore region of the channel) and detected with an anti-KV10.1 polyclonal antibody. C, KV10.1 immunostaining using mAb33 (upper panel) and mAb62 (lower panel) for IGR39 (left) and IPC298 (right) melanoma cell lines. D, KV10.1 currents elicited in HEK293-KV10.1 (left traces), IPC298 (middle traces), and IGR39 (right traces) cell lines. HEK293-KV10.1 (n = 21), IPC298 (n = 82), and IGR39 (n = 121). E, reduction on KV10.1 currents by 5 μm astemizole. Current amplitudes were determined as mean current in the last 100 ms of a 1.3-s depolarization to +60 mV for 9 (IPC298, left) and 10 cells (IGR39, right) F, reduction of KV10.1 currents by a KV10.1 siRNA. Cells were transfected with siRNA for KV10.1 and currents measured after 30 h, as described for E. The graphs for IPC298 (left panel) (scrambled n = 8, siRNA n = 11) and IGR39 (right panel) (scrambled n = 9, siRNA n = 10) show that more than 80% of the current was blocked by the siRNA against KV10.1. ****, p < 0.0001.
	FIGURE 2. Detection of KV10.1 splice variants in human cell lines and brain tissue. A, 1.5% agarose gel with nested PCR products obtained with the specific primers for KV10.1 listed in Table 1. Lanes 1 and 2, cDNAs for IPC298 and IGR39 cells, respectively; lane 3, blank. B, scheme of the exon structure of KV10.1 (E100) and the splice variants E65 and E70. C, Northern blot analysis of variant mRNA expression in human tissues and human cells. 30 μg of mRNA from the cell lines and tissues indicated was separated in a 2% formaldehyde agarose gel, transferred to Hybond N membranes, and hybridized overnight to a 700-bp DNA probe for the C terminus of KV10.1 channel. D, detection of E65 and E70 splice variant by ribonuclease protection assay. Two antisense 32P-labeled RNA probes (342 bp for E65 and 197 bp for E70) were hybridized with mRNA isolated from tumor cells. The probes protected from RNase digestions were analyzed by polyacrylamide gel electrophoresis and autoradiography. The 342-bp (left panel) and the 197-bp (right panel) protected fragment represent the 3–10 and the 3–8 exon junctions, respectively. E, 5′-RACE (left panel) and 3′-UTR amplification (right panel) in human brain tissue. The amplified products from RACE amplifications and 3′-UTR nested PCR were separated in 1,5% agarose gel.
	FIGURE 3. E65 and E70 co-precipitate with E100. A, right panel, immunoprecipitation of mVenus isoforms with anti-GFP antibody co-precipitated E100 protein of ∼130 and ∼110 kDa in E100 + E65 samples and E100 protein of ∼110 and ∼100 kDa in E100 + E70 samples. Left panel, input samples consisting of 10% (20 μg) not immunoprecipitated cell extracts. B, right panel, immunoprecipitation of E100 with a KV10.1 transmembrane domain-targeting antibody co-precipitated E65 and E70 proteins of ∼65 and ∼70 kDa in E100 + E65 and E100 + E70 samples, respectively. Left panel, input samples consisting of 10% (20 μg) non-immunoprecipitated cell extracts. As a detection antibody anti-GFP was used in both the left and the right panels. WB, Western blot.
	FIGURE 4. Analysis of E100 glycosylation upon co-expression with E65 and E70. A, co-immunoprecipitation with an anti-KV10.1 monoclonal antibody (mAb33) of E100 with the short isoforms E65 and E70 was performed, and precipitates were digested using Endo H and PNGase F to highlight the E100 glycosylation pattern. Notice that E130 is virtually absent when E100 is co-expressed with E70. B, immunoprecipitation of E100 and E70 proteins in oocytes injected with increasing ratios of E70/E100. The E110/E130 ratio increases together with increasing ratios of E70/full-length channel cRNA injected. C, densitometry analysis of data presented in Fig. B. D, immunoprecipitation of E100 with the short isoform E65 performed in oocyte lysates (15) using an anti-KV10.1 monoclonal antibody (mAb33) and detected with an anti- KV10.1 polyclonal antibody. Notice that E100 is strongly reduced when co-expressed with E65 at a 1:10 ratio. E, surface expression of the full-length form is not reduced by the spliced variants. Cells expressing a KV10.1 carrying an extracellular BTX binding site were labeled with biotinylated BTX, and the amount of label incorporated was quantified by sandwich ELISA (n = 9). WB, Western blot.
	FIGURE 5. KV10.1 current is down-regulated on co-expression with E65 and E70 in two different heterologous expression systems. A, E65 or E70 alone fails to generate potassium currents in oocytes. Traces were obtained from oocytes injected with E70, E65, or uninjected. None of the conditions gave rise to significant potassium currents. B, representative current traces for full-length KV10.1 (E100, left panel), E100 + E65 (middle panel), and E100 + E70 (right panel) from voltage clamp experiments in Xenopus oocytes. C, current-voltage relationship for E100 (black circles, n = 20), E100 + E65 (red squares, n = 16), and E100 + E70 (blue triangles, n = 20). D, current traces obtained from oocytes expressing KV1.4 either alone or together with E65 or E70. Current kinetics was not altered by co-expression. E, current-voltage relationship in oocytes expressing KV1.4 together with E65 or E70 1:10. E70 had no detectable effects on the current (n = 24), whereas E65 induced a modest reduction (37 ± 16%, p = 0.02 at +80 mV, n = 18). The effects of E65 alone on the properties of the oocytes could be responsible in part for this phenomenon. F, representative traces from patch clamp experiments with HEK cells stably expressing E100 and transfected with mVenus (left panel), E65mVenus (middle panel), or E70mVenus (right panel). Only green fluorescent cells visualized under an epifluorescence microscopy were used for recordings. G, current-voltage relationship for HEK-KV10.1 (black circles, E100, n = 12), E100 + E70 (blue triangles, n = 11), and E100 + E65 (red squares, n = 13). *, p < 0.05; **, p < 0.01; ***, p < 0.001. H, representative plots of variance versus mean current for KV10.1 (black), KV10.1 + E65 (red), and KV10.1 + E70 (blue), with both E65 and E70 injected at a 1:10 ratio. The parameters resulting in the fit shown are detailed in “Results.”
	FIGURE 6. TCC is not necessary for the interaction between KV10.1 and the splice variant E70 but impairs the current reduction induced by E65. A, immunoprecipitation of mVenus-fused splice variants E65L405Y and E70L556Y transiently expressed in HEK cells with anti-GFP antibody co-precipitated KV10.1 protein (∼130 kDa). Input, 10% (20 μg) of cell extracts. B, patch clamp recordings of HEK cells, with typical current traces from transfected cells co-expressing mVenus (left panel), E100 + E65L405YmVenus (middle panel), or E100+E70L556YmVenus (right panel). Only green fluorescent cells visualized under an epifluorescence microscope were used for recordings. C, I-V graph representing the mean current density at each voltage step for HEK- KV10.1 transfected with mVenus (black circles), E65L405YmVenus (red squares), and E70L556YmVenus (blue triangles). *, p < 0.05; ***, p < 0.001).
	FIGURE 7. Expression of E65 in Xenopus oocytes induces oocyte maturation independently of cyclin B and increases Cdc2 kinase activity. A, I-V relationship corresponding to the current for E100 (black), E100 + E65 in a 1:10 ratio (red), and E100 + progesterone pulse (open circles). ***, p < 0.001; asterisks apply for the full-length KV10.1 + E65 and also the progesterone pulse. B, GVBD was visible in oocytes injected with RNase-treated extract from E65-expressing oocytes (right panel) but not with RNase-treated extract from uninjected oocytes (left panel) or injected with extract from E70-injected oocytes (middle panel). C, extracts from naive (upper panel) or mature (progesterone-treated, lower panel) oocytes (n = 15) injected with the indicated cRNA combinations were immunoprecipitated and immunoblotted using an anti-cyclin B2 monoclonal antibody. D, Cdc2/Cdk1 activity was determined by ELISA under the described conditions. ****, p < 0.0001.

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