Figure 6.

14-3-3ζ interacts with and stabilizes TBC1D7. A, 293T cells were co-transfected with either vector and SBP-TBC1D7 or Myc-14-3-3ζ and SBP-TBC1D7 expression plasmids. Lysates were immunoprecipitated with Myc antibody. Immunoprecipitates (IP) and input lysates (WCL) were resolved on SDS-PAGE and blotted with SBP, Myc, and β-actin antibodies. B, 293T cells were transfected with either vector or Myc-14-3-3ζ. Whole-cell lysates were resolved on SDS-PAGE and blotted with TBC1D7, Myc, and β-actin antibodies. Relative expression of endogenous TBC1D7 is indicated, as determined by densitometric analysis. Values were normalized to β-actin loading control. The expression level of endogenous TBC1D7 from vector-transfected control cells was set to 1. C, 293T cells were co-transfected with Myc-14-3-3ζ and SBP vector, SBP-TBC1D7-WT, or SBP-TBC1D7-S124A. Lysates were subject to pulldown analysis with streptavidin beads. Affinity-purified complexes and input whole-cell lysates were resolved on SDS-PAGE and probed with Myc, SBP, and GAPDH antibodies. D, HeLa cells were transfected with either SBP vector or SBP-TBC1D7 expression plasmids. Cells were serum-starved (0% serum) overnight and treated for 15 min with 100 nm insulin or not. 10 μg of GSH Sepharose-conjugated recombinant GST-14-3-3ζ was incubated with cell lysates overnight. Co-purified complexes and input whole-cell lysates were resolved on SDS-PAGE and blotted with SBP, GST, P-Akt-Ser-473, and β-actin antibodies. The P-Akt-Ser-473 blot was stripped and reprobed with a pan-Akt antibody.