Fig. 3: SYVN1 ubiquitinates EGFR and enhances its stability.

A The EGFR protein was assessed in H1299 and PC9 cells overexpressing SYVN1. B The EGFR protein was assessed in H1299 and H1975 cells expressing shSYVN1. C, D The EGFR protein was detected in H1299 cells with SYVN1 overexpression (C) or knockdown (D) under conditions with or without EGF (100 ng/mL) stimulation for 10 min. E, F H1299 cells were treated with CHX (50 μg/mL) for various times, and the degradation rate of EGFR protein in H1299 cells overexpressing SYVN1 under conditions with (E) or without (F) serum was evaluated. G, H H1299 cells overexpressing (G) or knocking down (H) SYVN1 were treated with MG132 (20 μM) or CQ (100 μM) for 6 h, and the EGFR protein levels were detected by Western blot. I HEK293T cells were co-transfected with HA-SYVN1 (WT or C329S) and Flag-EGFR plasmids. Immunoprecipitation was performed to enrich Flag-EGFR, followed by assessment of its ubiquitination levels. J The EGFR protein was immunoprecipitated in H1299 cells overexpressing SYVN1 (WT or C329S), and its ubiquitination levels were assessed by Western blot. K The EGFR protein was immunoprecipitated in H1299 cells with SYVN1 knocked down, and its ubiquitination levels were assessed by Western blot. L In vitro ubiquitination assays were performed using purified SYVN1 and EGFR. M HEK293T cells were co-transfected with His-SYVN1, Flag-EGFR and various HA-Ub (WT, K48, K63, K48R, or K63R) plasmids. Immunoprecipitation was performed to enrich Flag-EGFR, followed by assessment of its ubiquitination levels by Western blot. All experiments were conducted independently three times. E, F Data are represented as the mean ± SD. P values were determined by independent samples t test, *p < 0.05; **p < 0.01; ***p < 0.001.