FIG. 1.

Receptor internalization does not require PKB/Akt. NIH 3T3 fibroblasts were transfected with human αvβ3 (hαvβ3) (A and E), hα5β1 (B and F), or hTfn-R (C and G) together with wt-PKB or PKB-AAA. Cells were serum starved for 30 min at 37°C with the inclusion of 100 nM wortmannin (WMN) or dimethyl sulfoxide (DMSO) for the last 10 min of the starvation period and then surface labeled with 0.2 mg of N-hydroxysuccinimide-S-S-biotin/ml at 4°C for 30 min. Internalization was determined by warming the cells to 37°C for the times indicated in the presence of 0.6 mM primaquine. Biotin was released from proteins remaining at the cell surface by incubation with β-mercaptoethanesulfonic acid (MESNa), and biotinylated proteins were determined by capture ELISA with microtiter wells coated with monoclonal antibodies against hβ3 (A and E) and hα5 (B and F) integrins or human CD71 (C and G). Values are means ± standard errors of the means (n > 4). For analysis of cellular phospho-GSK-3 levels, cells were transfected with wt-PKB or PKB-AAA by using the Amaxa Nucleofector. Cells were then serum starved and incubated in the presence and absence of 10 ng of PDGF-BB/ml for 10 min. Cell lysates were then probed for the presence of GSK-3β phosphorylated at Ser⁹ and total GSK-3β by Western blotting (D).