FIGURE 6.

IBtkα promotes the Pdcd4 proteasomal degradation upon serum stimulation. A, serum deprivation does not affect the IBtkα-dependent Pdcd4 degradation. HeLa cells (3 × 10⁶) were transfected with IBtk siRNA or control siRNA (siRNA-NT). Forty-eight hours later, cells were incubated with CHX (100 μg/ml) and serum-starved for up to 5 h. Cell lysates (30 μg) were separated by 4–12% NuPAGE and analyzed by WB with the indicated antibodies. B, IBtkα RNA interference counteracts the serum-induced Pdcd4 degradation in starved cells. HeLa cells (3 × 10⁶) were transfected with IBtk siRNA, control siRNA, or left untransfected (mock). 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. The following steps were as described in A. C, quantification of Pdcd4 half-life with or without IBtkα RNA interference upon serum addition in starved cells. Protein band intensities of the experiment described in B 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. D, overexpression of IBtkα augments the degradation of Pdcd4. HeLa cells (3 × 10⁶) were transfected with two amounts (4 or 8 μg) of IBtkα-FLAG or deletion mutants or empty vector. Forty-eight hours later, cell lysates (30 μg) were separated by NuPAGE 4–12% and analyzed by WB with the indicated antibodies. E, quantification of the Pdcd4 level of the experiment described in D. Protein bands were normalized to the corresponding GAPDH intensity. The mean densitometric values ± S.D. of three independent experiments are shown. F, MG132 proteasome inhibitor rescues Pdcd4 from IBtkα-mediated degradation. HeLa cells (3 × 10⁶) were transfected with IBtkα-FLAG or empty vector (4 μg) and 48 h later were serum-starved for 16 h and then replenished with serum (10%) and incubated with MG132 (20 μm) for up to 5 h. G, Cul3 RNA interference counteracts the serum-induced Pdcd4 degradation. HeLa cells (3 × 10⁶) were transfected with Cul3 siRNA or control siRNA and subjected to serum starvation/addition and CHX treatment, as detailed in B. Cell extracts were analyzed by WB with the indicated antibodies. H, quantification of Pdcd4 half-life of the experiment described in G. Protein bands were normalized to the corresponding GAPDH intensity and then compared with the 0 h time point. The mean densitometric values ± S.D. of three independent experiments are shown.