Fig. 4: BECN1 Y333 phosphorylation is required for IL-6-induced autophagy.
From: IL-6 regulates autophagy and chemotherapy resistance by promoting BECN1 phosphorylation
a IP analyses for VPS34, VPS15, BCL2, Rubicon, UVRAG, ATG14, and BECN1 in HEK293T cells in the absence or presence of IL-6 (20 ng/ml) for 12 h or in the absence or presence of CHZ868 (0.2 μM) treatment for 12 h. Western blotting was performed on WCEs (lower panels). b Co-IP analyses for HA-BECN1, VPS15, BCL2, Rubicon, UVRAG, ATG14, and VPS34 in HEK293T cells expressing HA-BECN1 WT, HA-BECN1 Y333F, or HA-BECN1 Y333E. Western blotting was performed on whole-cell lysates (lower panels). c Western blot analyses for BECN1, SQSTM1, and LC3B-II in MCF-7 cells expressing a vector control (Vector), HA-BECN1 WT, HA-BECN1 Y333F, or HA-BECN1 Y333E in the absence or presence of IL-6 (20 ng/ml) for 12 h. d Western blot analyses for BECN1, SQSTM1, LC3B-II in LoVo-BECN1-KO cells expressing a vector control (Vector), HA-BECN1 WT, HA-BECN1 Y333F, or HA-BECN1 Y333E in the absence or presence of IL-6 (20 ng/ml) for 12 h. e, f IF analyses for GFP-LC3B3 puncta in LoVo-BECN1-KO cells expressing a vector control (Vector), HA-BECN1 WT, HA-BECN1 Y333F or HA-BECN1 Y333E in the absence or presence of IL-6 (20 ng/ml) for 12 h. Confocal microscopy images are shown (n = 3 independent experiments) (f). Quantification of the number of GFP-LC3B puncta is shown in the right panel (g). *P < 0.05, ***P < 0.001. NS not significant. Scale bars, 20 μm. The values are presented as the mean ± s.d.; p values (Student’s t test, two-sided) with comparisons made to the control or different indicated groups are shown. Western blots are representative of two independent experiments. WCE whole-cell extract. Source data are provided in the Source Data file.
