siRNA transfection and rescue experiments

siRNAs (individual, ON-TARGET plus or siGENOME grade) were purchased from Dharmacon as follows: μ2: 5′-AAG​UGG​AUG​CCU​UUC​GGG​UCA-3′ (Huang et al., 2004); σ2 targeting 3′UTR: 5′-CCG​UGU​GUG​UCC​CGA​GUA​A-3′ (J-011833-09-0005); CHC: D-004001-02 (Huang et al., 2004); Grb2: 5′-CAU​GUU​UCC​CCG​CAA​UUA​UUU​UU-3′ (Jiang et al., 2003); Cbl-b: 5′-GGA​CAG​ACG​AAA​UCU​CAC​AUU-3′ (Huang et al., 2006); and c-Cbl: 5′-CCU​CUC​UUC​CAA​GCA​CUG​AUU-3′ (Huang et al., 2006).

The specificity of siRNAs (except of siRNA to σ2) was validated previously (Huang et al., 2004; Jiang et al., 2003). HeLa/FAP-EGFR cells were reverse-transfected in 6-well dishes with 50 nM siRNA (final concentration) and 3.5 µl DharmaFECT-1 in 1 ml of complete media without antibiotics. 2 d later, cells were split and plated on 96-well plates, coverslips, and other dishes (for Western blot analysis). The next day, the cells were starved overnight and used for experiments on day 4 after siRNA transfection. In AP2σ-GFP rescue experiments, cells were replated into 12-well dishes 36 h after siRNA transfection and transfected with DNA plasmids 12 h after plating. The next day, the cells were split again and plated onto 96-well plates or coverslips, serum-starved overnight, and used for experiments on day 5 after siRNA transfection. PAE cells were transfected with 50 nM μ2 siRNA 24 h after plating. 2 d later, the cells were replated and 8 h later were again siRNA-transfected. 2 d later, the cells were replated and used for experiments 24 h later.