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. Author manuscript; available in PMC: 2016 Jul 19.
Published in final edited form as: Biochemistry. 2015 Apr 6;54(15):2473–2480. doi: 10.1021/acs.biochem.5b00016

Figure 3.

Figure 3

IRS2 protein level and Plk1-dependent phosphorylation are cell cycle regulated. A, Mitotic specific phosphorylation of IRS2 at S556 and S1098. Exponentially growing or mitosis enriched (via nocodazole treatment) - HeLa cells were harvested for IB against two phospho-specific antibodies and IRS2. B, HeLa cells were arrested at the G1/S boundary using a double thymidine block (DTB) protocol, released for different times and harvested for IB. C, Dephosphorylation of S556 and S1098 of IRS2 during mitotic exit. HeLa cells were arrested at mitosis with nocodazole, released for different times and harvested. D-E, Co-localization of Plk1 with phospho-S1098-IRS2 at mitotic structures. HeLa cells were cultured on coverslips and subjected to immunofluorescence (IF) staining with antibodies against Plk1 and IRS2 (D) or pS1098-IRS2 (E). DNA was stained with DAPI. Arrows showing the co-localization of Plk1 and pS1098-IRS2 are indicated in each panel. F, Co-localization of Plk1 with phospho-S1098-IRS2 at spindle polea. Cells were pre-treated with 0.25% TritonX100 for 2 min, followed by IF as in E. G, Overexpression of IRS2-WT does not affect mitotic exit. HeLa cells were transfected with IRS2-myc, treated with nocodazole and released for different times for IB. H, Depletion of IRS2 does not slow down mitotic exit. HeLa cells were transfected with IRS2 shRNA and processed as in G.