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. Author manuscript; available in PMC: 2021 Feb 24.
Published in final edited form as: Nat Cell Biol. 2020 Aug 24;22(9):1064–1075. doi: 10.1038/s41556-020-0562-4

Figure 1 |. PD-L1 is acetylated at the lysine 263 residue by p300.

Figure 1 |

a. Immunoblot (IB) analysis of whole-cell lysates (WCL) and anti-PD-L1 immunoprecipitates (IPs) derived from MDA-MB-231 cells. Immunoglobulin G (IgG) served as a negative control. b. IB analysis of WCL and anti-Myc IPs derived from 293T cells transfected with Myc-PD-L1 and HA-tagged p300, GCN5, PCAF, Tip60 or CBP. c. IB analysis of WCL and anti-HA IPs derived from MDA-MB-231 PD-L1 knockout (KO) cells re-introduced HA-ins-PD-L1 (HA-tag was inserted following the signal peptide) and treated with DMSO or indicated concentration of A485 for 4 hrs. d. IB analysis of WCL and anti-PD-L1 IPs derived from MDA-MB-231 cells transduced with shRNAs against p300 or shGFP as negative control. e. IB analysis of WCL and anti-PD-L1 IPs derived from MDA-MB-231 cells transduced with shRNAs against CBP or shGFP as negative control. f. In vitro acetylation assay using purified His-PD-L1 recombinant protein incubated with p300 in the presence or absence of Acetyl-CoA. g. A schematic illustration of PD-L1 protein domains and amino acid residues in the cytoplasmic domain (C-tail). SP, signal peptide; TM, transmembrane domain. h. IB analysis of WCL and anti-HA IPs derived from 293T cells transfected with Myc-p300 and HA-full length (FL) PD-L1 or the deletion mutant of C-tail (263–290 a.a.). i. IB analysis of WCL and GST pull-down products derived from 293T cells transfected with Myc-p300 and GST-C-tail PD-L1 or KR mutants.

The western blots in a-f, h and i were performed for n=2 independent experiments with similar results. Unprocessed immunoblots are shown in Source Data Fig. 1.