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. 2021 Jul 27;12:4536. doi: 10.1038/s41467-021-24769-3

Fig. 6. NEK2 phosphorylates PD-L1 at T194/T210 to maintain protein stability.

Fig. 6

a Schematic diagram of the NEK-binding motif (F/LXXS/T) in the glycosylation-rich region and amino acid sequences around the potential binding sites of PD-L1 were aligned in evolutionarily divergent species. The F/LXXS/T motifs are highlighted in blue. b Generation of site-specific antibodies against T194 and T210 (Mus musculus: T193 and T209)-phosphorylated PD-L1. c Western blot analysis of T193 and T209-phosphorylated PD-L1 in KPC cells with NEK2 inhibitor (10 μM, 24 h) and NEK2 KD KPC. Representative image is shown n = 3 independent experiments. d In vitro kinase assay and western blot analysis of pT193-PD-L1 and pT209-PD-L1 expression of recombinant PD-L1 WT and NEK2 (active) protein. Representative image is shown, n = 3 independent experiments. e, f Western blot analysis of PD-L1 expression in flag-PD-L1 WT and T193/209A or T193/209D-transfected KPC cells with or without MG132 treatment. Representative image is shown n = 3 independent experiments. g Western blot analysis of pT193-PD-L1, pT209-PD-L1, and PD-L1 expression in WT and K37R transfected NEK2 KD KPC cells. Representative image is shown n = 3 independent experiments. h Ubiquitination assay of PD-L1 in Flag-PD-L1 WT and T193/209A or T193/209D-transfected KPC cells, subjected to anti-PD-L1 IP and anti-ubiquitin Western blot analysis after treatment with MG132 (50 μM, 24 h). Representative image is shown n = 3 independent experiments. i Ubiquitination assay of PD-L1 in WT and K37R transfected NEK2 KD-KPC cells subjected to PD-L1 IP and Western blot analysis with anti-ubiquitin after treatment with MG132 (50 μM, 24 h). Representative image is shown n = 3 independent experiments.