FIGURE 7.

IBtkα-mediated ubiquitylation of Pdcd4 does not depend on serines 71 and 76. A, overexpression of IBtkα induces the degradation of Pdcd4 S67A/S71A/S76A-HA mutant. HeLa cells (3 × 10⁶) cells were transfected with Pdcd4-WT-HA (2 μg) or Pdcd4 S67A/S71A/S76A-HA (2 μg) expression vectors, with or without IBtkα-FLAG (4 μg) or empty (4 μg) vector. Forty-eight hours later, cells were serum-starved for 16 h (time point 0) and then replenished with serum (10%) and incubated with CHX (100 μg/ml) for up to 5 h. Cell lysates (30 μg) were separated by NuPAGE 4–12% and analyzed by WB with the indicated antibodies. B and C, quantification of Pdcd4-WT-HA (B) or Pdcd4 S67A/S71A/S76A-HA (C) half-life with or without IBtkα overexpression upon serum addition in starved cells. Protein band intensities of the experiment described in A were normalized to the corresponding GAPDH intensity and then compared with the 0 h time point. The mean densitometric values ± S.D. (error bars) of three independent experiments are shown.