Fig. 3
LNK suppresses signaling of the IFN-STAT pathway. a Western blots show forced expression of LNK suppress the IFN-γ (2000 U/ml, 24 h) induced STAT1 phosphorylation in M202 cell. Cells were growth in either normal complete media (with 10% FBS) or serum starved (24 h) before IFN-γ treatment. GFP, GFP overexpressing control; LNK, LNK overexpressing cells; C-PARP, cleavage PARP; Exp, exposure time. b Western blots show silencing of LNK enhances the IFN-γ (2000 U/ml, 24 h) induced STAT1 phosphorylation and enhanced expression of downstream markers (IRF-1 and PD-L1) in M229 cell. Cell were grown in either normal complete (with 10% FBS) or serum starved media 24 h before the IFN-γ treatment. Ctrl, control; sgLNK, CRISPR-Cas9 guide RNA targeting LNK (sgLNK-1). c Western blots showed that forced expression of LNK suppresses the IFN-γ induced STAT1 phosphorylation and IRF1 expression in A375 cells (left panel), while silencing LNK generated the opposite effect. Ctrl, non-target shRNA control; shLNK, shRNA targeting LNK (shLNK-16). d Western blots show force expression of LNK suppresses interferon alpha (IFNA1 as well as IFNA2B) or interferon beta (B1A, Interferon beta 1A; B1B, Interferon beta 1B) induced STAT1 phosphorylation in M202 cell. e Immunoprecipitation (IP) experiments showed that LNK interacted with STAT1 protein. Melanoma cells were treated with IFN gamma (2000 U/ml) for 30 min. Lysates were extracted (m-PER protein extraction solution) and proteins were pull-downed using LNK (Santa Cruz Biotechnology (A-12): sc-393709) or STAT1 (Cell Signaling, 9172) antibody. f GST-pull down experiments showed that the PH and SH2 domains of LNK bind to STAT1 protein. GST-PH-LNK, GST protein fused to the PH domain of LNK; GST-SH2-LNK, GST protein fused to the SH2 domain of LNK. g GST-pull down experiments showed the GST-STAT1 protein (expressed from E.coli) directly binds to the LNK protein (V5 tag)
