Figure 5
Simultaneous depletion of GGA1 or GGA3 with GGA2 restores EGFR expression. (a and b) ARPE-19 cells were transfected with control (siCtrl), GGA1 (siGGA1), GGA2 (siGGA2), or GGA3 (siGGA3) siRNAs alone, or with a combination of siGGA2 and siGGA1, or siGGA2 and siGGA3 as indicated. Cells were then fixed for immunofluorescence staining with anti-EGFR antibody (a), or were lysed for western blot analysis with antibodies against EGFR, GGA1, GGA2, GGA3, and GAPDH (b). Nuclei were labeled with Hoechst 33342. Bar, 20 μm. (c) Cell lysates from HEK293 cells expressing GFP-jxt were pulled down using GST fusion proteins containing wild type VHS-GAT (WT) of GGAs or mutant forms (N92A, N108A, or N91A) of VHS-GAT derived from GGA1 (GST-1VG), GGA2 (GST-2VG), or GGA3 (GST-3VG). Bound proteins were immunoblotted using anti-GFP antibody. Ponceau staining is shown at the bottom. Uncropped blots are presented in Supplementary Fig. 7. (d) ARPE-19 cells transfected with siCtrl, siGGA1, or siGGA3 were used for PLA to detect GGA2-EGFR interaction (green). Nuclei were stained with DAPI (blue). Bar, 20 μm. (e) PLA signals/cell were counted and mean values ± SD (n = 575 [siCtrl], n = 482 [siGGA1], and n = 448 [siGGA3]) were plotted. Differences were analyzed by one-way ANOVA (P < 0.001) and Tukey’s honestly significant difference test (***P < 0.001, ns: not significant).
