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. 2022 Jan 31;12(5):2080–2094. doi: 10.7150/thno.69444

Figure 4.

Figure 4

Phosphorylation at serine-311 of p65 impedes HDAC5-p65 interaction. (A) PANC-1 cells were infected with indicated plasmid for 24 h and treated with mock treatment (DMSO) or TNF-α for another 24 h. Then cells were harvested for co-IP assay. (B) WCL from PANC-1 cells were treated with or without λ-phosphatase, and then were applied to co-IP assay. (C) Western blot analysis of p65 protein from WCL of PANC-1 cells pulled down by GST or GST-HDAC5-DAC under the condition with or without λ-phosphatase treatment. (D) Schematic diagram depicting putative phosphorylation site on the C terminal of p65. (E) Western blot analysis of HDAC5 from WCL of PANC-1 pulled down by GST or indicated mutated GST-p65-C recombinant proteins. (F-H) PANC-1 cells were transfected with indicated plasmid for 48 h, and were harvested for co-IP assay (F), RT-qPCR (g) and ChIP qPCR (H). Data are shown as mean ± SD (n = 3, n.s. not significant, * P < 0.05, ** P < 0.01, *** P < 0.001). (I) PANC-1 cells were treated with indicated drugs (Staurosporine, 1 µM) or mock treatment (DMSO) for 24 h, and were harvested for co-IP assay. (J) Model depicting the mechanism that phosphorylation on S311 of p65 impedes HDAC5-p65 interaction and promote PD-L1 transcription.