Fig. 2

Cdc42 required for LAPTM4B-35 surface localization. (A), LAPTM4B KO cells stably expressing Flag-tagged LAPTM4B-35 or LAPTM4B-24 were transfected with constitutively active Cdc42^G12V-GFP. The Cdc42^G12V-GFP was pulled down using GFP-trap, with coimmunoprecipitated LAPTM4B, and then detected by Western blotting. The blot was reprobed with GFP antibody. Left panel: representative experiment. Right panel: quantification of n = 3 experiments, mean ± SEM, data normalized to ?L4B-35,? p = 9.87 × 10^?5. ?Lysate? denotes total cell lysate, ?IP? the immunoprecipitated protein, and ?post IP? the remaining supernatant after immunoprecipitation. (B), Cells stably expressing LAPTM4B-35 and LAPTM4B-24were transfected with the dominant negative Cdc^T17N-GFP. The Cdc42^T17N-GFP was pulled down using GFP-trap, and its interaction with the LAPTM4B isoforms was assessed by Western blotting. Left panel: a representative experiment. Right panel: quantification of three experiments, mean ± SEM, data normalized to ?L4B-35.? p = 0.0003. (C), Coimmunoprecipitation of endogenous LAPTM4B with Cdc^G12V-GFP from WT A431 cells. LAPTM4B KO cells served as a control for antibody specificity. The Cdc42-GFP was pulled down using GFP-trap, and the interaction was further assessed by Western blotting using anti-LAPTM4B antibody. Left panel: a representative experiment. Right panel: quantification of three experiments, mean ± SEM, p = 5.09 × 10^?5. D, Whole-cell lysates were assessed for active Cdc42. Lysates treated with GTP?S or with GDP served as the positive and negative control. E, Plasma-membrane expression of LAPTM4B-35 was assessed by surface biotinylation in cells stably expressing LAPTM4B-35-Flag, treated with control, Cdc42, or LAPTM4B siRNAs, followed by Western blotting using anti-Flag antibody. Left panel: representative experiment. Right panel: quantification of three experiments, mean ± SEM, data normalized to ?Ctrl siRNA,? p = 0.0009