Wong OK , Fang G .

GM062852 NIGMS NIH HHS , R01 GM062852 NIGMS NIH HHS

	Figure 1. The 3F3/2 epitope in X. laevis is a phosphospecific kinetochore antigen. (A) Asynchronous XTC cells in mitosis (M) and in interphase (I) were stained with the 3F3/2 antibody and with an anti–xCenp-A antibody. The majority of the xCenp-A dots in M cells contained the 3F3/2 signals, although a few lacked the 3F3/2 staining, likely because of the dephosphorylation that occurred during sample processing. (B and C) XTC cells arrested with taxol or released from taxol arrest were stained for the 3F3/2 antigen and for xCenp-A or xBub1. Insets show a magnified image of the boxed areas. (D) Nuclei purified from checkpoint extracts (top) were dephosphorylated with λ-phosphatase (middle) and then rephosphorylated with ATP (bottom). (A–D) Red, 3F3/2; green, xCenp-A or xBub1. Arrowheads and arrow point to the 3F3/2 signals on centrosomes and at the midbody, respectively. Bars, 5 μm.
	Figure 2. Plx1 is both necessary and sufficient to phosphorylate the 3F3/2 epitope at kinetochores. (A) Nuclei purified from checkpoint extracts carrying the 3F3/2 epitope (left) were dephosphorylated, treated with NEM, and rephosphorylated with either ATP (middle) or ATP plus CSF extract (right). Nuclei were then stained for the 3F3/2 epitope and xBub1. (B) Immunodepletion of kinetochore-associated kinases from CSF extracts. CSF extracts were depleted of xMps1 (lane 1), xBub1 (lane 4), xBubR1 (lane 8), and Plx1 (lane 11). 1 μl of depleted extracts and mock-depleted extracts (lanes 2, 5, 9, and 12) and 0.05 μl of input extracts (lanes 3, 6, 7, and 10) were analyzed by Western blotting to determine the depletion efficiency. xMad1 and xMad2 were shown here to demonstrate the specificity of the immunodepletion. (C and D) Rephosphorylation of 3F3/2 by extracts depleted of kinetochore-associated kinases. Nuclei were prepared as in A and rephosphorylated with either ATP (first column in C) or ATP plus depleted extracts prepared in B. ID, immunodepletion. (E) Mean kinetochore fluorescence intensity of xBub1 (green) and 3F3/2 (red) signals from samples rephosphorylated with xBubR1- or Plx1-depleted extracts. The fluorescence intensity was normalized to the corresponding values derived from mock-depleted extracts. Error bars represent SD. (F) Nuclei from checkpoint extracts were dephosphorylated and rephosphorylated with ATP or with ATP plus recombinant His6-Plx1. (A, C, D, and F) Red, 3F3/2; green, xBub1. Bars, 5 μm.
	Figure 3. Plx1 is the physiological 3F3/2 kinase. (A) Extracts were mock depleted (lane 6) or depleted of Plx1 (lanes 3–5). Plx1 (lane 4) or Plx1-KD (lane 5) was translated in CSF extracts that had been depleted of endogenous Plx1 and then added to the Plx1-depleted extracts. Lanes 1 and 2 show input extracts. The volumes of extracts loaded were as indicated. Different amounts of input extracts were loaded to quantify the degree of depletion and add-back. (B and D–F) 3F3/2, Plx1, xCenp-A, xMad2, and xBubR1 staining of nuclei purified from checkpoint extracts that had undergone immunodepletion (ID) and add-back (AB) of the indicated proteins. Red, 3F3/2; green, Plx1, xCenp-A, xMad2, and xBubR1. (C) Mean kinetochore fluorescence intensity (from 15 randomly selected kinetochores) of xBubR1 (green) and 3F3/2 (red) signals from samples that were depleted of Plx1 and then added back with the indicated proteins. The fluorescence intensity was normalized to the corresponding values derived from mock-depleted extracts. Error bars represent SD. Bars, 5 μm.
	Figure 4. Plk1 is required for the generation of the 3F3/2 epitope in HeLa cells. (A, top) Schematic of double-thymidine synchronization and siRNA transfection of HeLa cells. (bottom) Western blot analysis of Plk1 and control knockdown cell lysates to determine the knockdown efficiency. p38 MAPK was used as a loading control. The volumes of HeLa cell lysates loaded were as indicated. Different amounts of cell lysates were loaded from the control knockdown sample to quantify the degree of Plk1 knockdown. (B and D–G) Plk1 or control knockdown cells were fixed at 11 h after release from the second thymindine arrest. Prometaphase cells were stained with the following antibodies: (B) Crest serum (red) and anti-Plk1 (green); (D) 3F3/2 antibody (red) and anti-Plk1 (green); (E) 3F3/2 antibody (red), Crest serum (green), and anti-Plk1 (green); (F) Plk1 (red) and Mad2 (green); (G) Plk1 (red) and BubR1 (green). In D, F, and G, arrowheads point to 3F3/2 and Plk1 signals at spindle poles. Bars, 5 μm. (C) Mean kinetochore fluorescence intensity (from randomly selected kinetochores of multiple prometaphase cells) of Plk1, 3F3/2, Mad2, and BubR1 signals from Plk1 (green) or control knockdown (red) cells. The fluorescence intensity was normalized to the corresponding values derived from control knockdown cells. Error bars represent SD. (H) HeLa cells were synchronized by double-thymidine arrest/release and transfected with siRNAs as described in A, except that transfected cells were released from the second thymidine arrest in either the presence (+ Noc) or absence (− Noc) of 100 ng/ml nocodazole. At 14 h after release from the second thymidine arrest, cells were fixed, and the mitotic index was counted (n > 150 cells for each sample).
	Figure 5. xMps1 controls the kinetochore localization of Plx1. (A) CSF extracts were either mock depleted or depleted of xMps1 and then added back with the indicated proteins. Nuclei were purified from depleted checkpoint extracts and stained for xMps1, Plx1, and xCenp-A. ID, immunodepletion; AB, add-back. Bar, 5 μm. (B) Depletion efficiency was determined by Western blot analysis of equal volumes of mock- (lane 1) or xMps1-depleted (lane 2) extracts as well as xMps1-depleted extracts with the add-back of xMps1 (lane 3) or xMps1-KD (lane 4).
	Figure 6. xMps1 and Plx1 are required for the assembly of the outer kinetochore structure. (A) Nuclei were purified from mock-, xMps1-, or xMps1-depleted extracts with the add-back of the indicated proteins, as described in Fig. 5. Purified nuclei were stained for xNdc80 or xNuf2. (B) Nuclei were purified from mock-, Plx1-, or Plx1-depleted extracts with the add-back of the indicated proteins, as described in Fig. 3. Purified nuclei were stained for xNdc80 and xNuf2. ID, immunodepletion; AB, add-back. Bars, 5 μm.

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