Figure 2. SNX12 overexpression inhibits EGFR transport and degradation without affecting retrograde and recycling transport routes.

(A-C) After cell surface binding, EGF-biotin coupled to streptavidin^{AlexaFluor488} was endocytosed for 10 min (A) or 50 min (B-C) at 37°C in HeLa cells expressing mRFP1-SNX12. Cells were labeled with anti-EEA1 (A) or Lamp1 (B-C) antibodies and analyzed by triple channel fluorescence. (D) HeLa cells expressing myc-SNX12 or myc-SNX3 or mock-treated were incubated with EGF for the indicated time periods. Cell lysates (100 µg) were analyzed by SDS gel electrophoresis and western blotting with antibodies against EGFR, α-tubulin (a-tub) or myc. (E) After cell surface binding, Shiga toxin B-subunit conjugated to Cy3 was internalized for 10 min or 50 min at 37°C into HeLa cells expressing GFP-SNX12. Cells were labeled with anti-transferrin receptor (TfR) or Rab6 antibodies and analyzed by triple channel fluorescence. (F) After cell surface binding (0 min), transferrin conjugated to AlexaFluor546 (transferrin⁵⁴⁶) was internalized for 30 min or 120 min at 37°C into control cells (upper panels) or cells expressing GFP-SNX12 (lower panels). Cells were then analyzed by fluorescence. (A-C and E-F) Scale bar indicates 10 µm.