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. 2017 Nov 18;8(62):105923–105935. doi: 10.18632/oncotarget.22510

Figure 1. USP18 interacted with IRS4.

Figure 1

(A) identification of USP18-binding proteins. 293T cell lines stably expressing Flag or Flag-USP18 were established. Cell protein lysates were precipitated with anti-Flag M2 affinity gel. The immunoprecipitated protein complexes were separated by SDS–PAGE, stained using Coomassie R-350 and analyzed by mass spectrometric analysis. (B) IRS4 interacted with USP18. 293T cells were co-transfected with Myc-IRS4 and Flag-USP18 or Flag empty plasmid. Cell lysates were immunoprecipitated (IP) with an anti-Flag antibody (incubated with anti-Flag M2 Affinity Gel) and analyzed by immunoblotting with anti-Myc (upper) and anti-Flag antibodies (lower). (C) Co-IP assay of the interaction between USP18 and endogenous IRS4. 293T cells were transfected with Flag or Flag-USP18. Cell protein lysates were immunoprecipitated with an anti-Flag antibody followed by immunoblotting with anti-IRS4(upper) and anti-Flag antibodies (lower). (D) Co-IP assay of the interaction between IRS4 and endogenous USP18. 293T cells were transfected with Flag or Flag-IRS4. Cell protein lysates were immunoprecipitated with an anti-Flag antibody followed by immunoblotting with anti-USP18(upper) and anti-Flag antibodies (lower). (E, F) IRS4 interacts with USP18 endogenously. 293T cells(E) and Huh7.5.1 cells(F) lysates were immunoprecipitated with an anti-IRS4 antibody or control IgG and analyzed by immunoblotting with anti-USP18 and anti-IRS4 antibodies. (G) IRS4 interacts with USP18 endogenously in JFH1-infected Huh7.5.1 cells. JFH1-infected Huh7.5.1 cells lysates were immunoprecipitated with an anti-IRS4 antibody or control IgG and analyzed by immunoblotting with anti-USP18, anti-IRS4 and anti-core antibodies.