Figure S2.

Efficiencies of siRNAs knockdowns and σ2-GFP assembly into AP2 in cells transfected with σ2 siRNA. (A–D) Typical efficiencies of protein depletion in siRNA experiments in HeLa/FAP-EGFR cells described in Figs. 1, 2, 7, and 9. HeLa/FAP-EGFR cells were transfected with nontargeting (NT) siRNA and Grb2 (A), c-Cbl and Cbl-b (A), CHC (B), μ2 (C), or σ2 (C and D) siRNAs. Cells were lysed 3–4 d after transfections, and lysates were blotted with indicated antibodies. β-Actin and Grb2 are loading controls. (E) HeLa/FAP-EGFR cells were transfected with σ2 siRNA for 2 d and then transfected with WT σ2-GFP plasmid. 2 d later, cells were lysed, and σ2-GFP was immunoprecipitated using the GFP antibody. Lysates (total cell lysates [TCL]) and immunoprecipitates (IP) were probed by Western blotting (WB) with GFP and α-adaptin antibodies to demonstrate the assembly of WT σ2-GFP into AP2 complexes. (F) PAE/EGFR-GFP cells were transfected with μ2 siRNAs as described in Materials and methods. Cells were lysed 5 d after two transfections, and lysates were blotted with α-adaptin and Grb2 (loading control) antibodies.