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. Author manuscript; available in PMC: 2019 Oct 10.
Published in final edited form as: Mol Cell. 2018 Aug 16;71(4):606–620.e7. doi: 10.1016/j.molcel.2018.07.030

Figure 4. Phosphorylation of S195 induces ER-associated degradation of PD-L1.

Figure 4.

(A) IP-MS analysis showing candidates with increased binding to PD-L1 S195E compared to PD-L1 S195A. ERAD components are shown in red. (B) IPA based on the mass data from panel (A) showing unfolding protein response and ubiquitination pathways closely related to ERAD were activated (C) MDA-MB-231 stable cells were treated with 50 μM cycloheximide (CHX) for the indicated time. The band intensity was quantified by Image J analysis. Data represent mean ± SD. n = 3. **P, 0.001~0.01, and NS, not significant, Student’s t test. (D) MDA-MB-231 stable cells were treated with proteasome inhibitor MG132 (10 μM) and ERAD inhibitor eeyarestatins (Eer I, 20 μM) for the indicated time. (E, F) MDA-MB-231 cells were treated with or without metformin (5 mM) for 24 hr and MG132 (10 μM) for 6 hr. Endogenous PD-L1 (E) or Flag-tagged WT, S195A, or S195E PD-L1 (F) was pulled down by the PD-L1 antibody (E) or Flag M2 magnetic bead (F), respectively, followed by Western blotting to detect ERAD components (SEL1L, HRD1, ERLEC1, and OS9). (G) MDA-MB-231 cells expressing exogenous HA-ubiquitin were cultured with or without metformin (5 mM) for 24 hr followed by MG132 (10 μM) for 6 hr. Ubiquitination of endogenous PD-L1 was examined by HA immunoblotting after IP with antibody against PD-L1. (H) Ubiquitination of WT, S195A, and S195E PD-L1 was examined by HA immunoblotting after IP with Flag M2 magnetic bead. (I) Control or HRD1 siRNA was transfected into MDA-MB-231 cells expressing exogenous HA-ubiquitin. Ubiquitination of endogenous PD-L1 in each transfectant was examined by HA immunoblotting after IP with PDL1 antibody. (J) Control or HRD1 siRNA was transfected into MDA-MB-231 WT and S195E PD-L1 stable cells expressing exogenous HA-ubiquitin. Ubiquitination of WT and S195E PD-L1 was examined by HA immunoblotting after IP with Flag M2 magnetic bead. (K) siRNA targeting SEL1L, HRD1, OS9 or ERLEC1 was transfected into MDA-MB-231 (L) Each siRNA for ERAD components was transfected into MDA-MB-231 S195E PD-L1 stable cells followed by immunoblotting with the indicated antibodies. M, mock. W, WT PD-L1. A, S195A PD-L1. E, S195E PD-L1.