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. 2012 Nov 12;288(1):455–465. doi: 10.1074/jbc.M112.397448

FIGURE 6.

FIGURE 6.

NRG blocks 14-3-3 binding-induced inhibition of SSH1L. A, MCF-7 cells were serum-starved for 16 h and then treated with NRG (100 ng/ml) for the indicated times. Endogenous SSH1L was immunoprecipitated (IP; anti-SSH1L antibody), and samples were subjected to SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting for binding to 14-3-3 (anti-14-3-3β antibody). Samples were also control-stained for SSH1L phosphorylation (anti-phospho-Ser-978 SSH1L antibody) and reprobed for SSH1L. B, MCF-7 cells were cotransfected with Myc-tagged SSH1L and vector control or constitutively active PKD1 (PKD1.CA) as indicated. SSH1L was immunoprecipitated (anti-Myc antibody), and samples were subjected to SDS-PAGE, transferred to nitrocellulose, and analyzed for binding of 14-3-3β (anti-14-3-3β antibody). For controls, samples were analyzed for phosphorylation of SSH1L at Ser-978 (anti-phospho-Ser-978 SSH1L antibody) and then reprobed for SSH1L (anti-Myc antibody). PKD1 expression was detected by Western blotting of lysates with anti-PKD1 antibody.