USP2 Inhibits Lung Cancer Pathogenesis by Reducing ARID2 Protein Degradation via Ubiquitination

Figure 3

USP2 modulated ARID2 protein level via ubiquitination suppression and subsequent ARID2 protein stabilization; H1299 cells were transfected with Sh USP2#1, Sh USP2#2, or negative control vector. A549 cells were transfected with USP2 overexpression vector or negative control vector. (a, b) CHX treatment of H1299 cell line groups transfected with USP2 shRNAs to evaluate the influences of USP2 expression suppression on ARID2 protein stability and degradation. Relative quantification of ARID2 protein level was analyzed in three groups treated by CHX for 0 h to 9 h ( indicates compared with negative control vector). (c, d) CHX treatment of A549 cell line groups transfected with USP2 or loss-of-function mutated USP2 overexpression vectors to evaluate the influences of USP2 overexpression on ARID2 protein stability and degradation. Relative quantification of ARID2 protein level was analyzed in three groups treated by CHX for 0 h to 9 h ( indicates compared with negative control vector; # indicates no statistical significance). (e) Ubiquitination analysis using the Co-IP method in H1299 cell line groups transfected with or without USP2 shRNAs. Anti-Poly-Ub antibody was used to detect ubiquitination levels in protein samples immunoprecipitated by the anti-ARID2 antibody. Whole cell lysate samples were used to detect USP2 and ARID2 protein levels. (f) Ubiquitination analysis using the Co-IP method in A549 cell line groups transfected with USP2 or mutated USP2 overexpression vectors. Anti-Poly-Ub antibody was used to detect ubiquitination levels in protein samples immunoprecipitated by the anti-ARID2 antibody. Whole cell lysate samples were used to detect USP2 and ARID2 protein levels. (g) Ubiquitination analysis using the Co-IP method in HEK-293T cell line groups transfected with His-ARID2 and Flag-Ub, in combination with different doses of HA-USP2. Anti-Poly-Ub antibody was used to detect ubiquitination levels in protein samples immunoprecipitated by the anti-His antibody. Whole cell lysate samples were used to detect HA-USP2 and His-ARID2 protein levels. (h) ARID2 ubiquitination analysis using the Co-IP method in HEK-293T cell line groups transfected with His-ARID2, in combination with HA-Vector, HA-USP2, or HA-mutated USP2, respectively. Anti-Ub antibody was used to detect ARID2 ubiquitination level in protein samples immunoprecipitated by anti-ARID2-(Ub) antibody. Whole cell lysate samples were used to detect HA-USP2 and His-ARID2 protein levels.

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