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. 2020 Jan 20;10:663. doi: 10.1038/s41598-019-57153-9

Figure 2.

Figure 2

Sym004 promotes ubiquitin-independent, ESCRT-dependent lysosomal degradation of EGFR. (a) SCC47 cells were serum-starved for 1 h, then MG132 (10 µM) or bafilomycin A1 (600 nM) were added, followed 1 h later by cisplatin (50 µM), cetuximab, Sym004 or EGF. The cells were incubated overnight and lysed with RIPA buffer. (b) SCC47 cells were serum-starved for 1 h, then bafilomycin A1 (600 nM) was added followed 1 h later by Sym004-AF568 or EGF-AF647. The cells were incubated overnight, fixed and stained with EGFR-AF488 antibody. (c) Serum-starved SCC47 were treated with cetuximab, Sym004 or EGF as indicated, then lysed. EGFR was immunoprecipitated with anti-EGFR antibody (normal mouse IgG used in Control). For quantifications, ubiquitylated EGFR was normalised to total EGFR. (d) Serum-starved, Hrs- or Tsg101-depleted (or control) SCC47 cells were treated with Sym004 (3 µg/ml or 30 µg/ml) or EGF for 6 h, then lysed. (e) The cells treated as in (d) were fixed and stained with EGFR-AF488. Nuclei were stained with Hoechst 33342. All immunoblots were cropped for clarity.