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. 2017 May 30;8:15427. doi: 10.1038/ncomms15427

Figure 3. PHB2 is activated by phosphorylation at Ser39 via PKCα.

Figure 3

(a) The inhibitory effects of siPKCα on PHB2 phosphorylation at S39 in PHB2 immunoprecipitates of cytoplasmic and nuclear fractions in MCF-7 cells. C-WCL, total cytoplasmic lysates; N-WCL, total nuclear lysates. (b) In vitro PKCα activation assay of PHB2 using engineered peptides representing S39 and the alanine mutant of S39 (S39A). These data represent the means±s.e.m. of three independent experiments. (c) Phosphorylation at S39 in the recombinant full-length PHB2 by PKCα using Phos-tag SDS–PAGE and western blot analysis with anti-PHB2 and anti-pS39-PHB2 antibodies. (d) Dephosphorylation of full-length PHB2 phosphorylation at S39 by PP1Cα using Phos-tag SDS–PAGE and western blot analysis with anti-PHB2 and anti-pS39-PHB2 antibodies. (e) Phosphatase activity of PP1Cα against purified, recombinant full-length PHB2 phosphorylation at S39. These data represent the means±s.e.m. of three independent experiments. (f) The effects of siPKA and siPKCα on BIG3–PHB2 complexes and PHB2 S39-phosphorylation in MCF-7 cells after E2 stimulation for 6 h (left), 12 h (middle) and 24 h (right). ***P<0.001 (two-sided Student’s t-test).