Fig. 5

USP15 regulates ERK2 ubiquitination and stimulates p-ERK1/2 levels. a ATDC5 cells were infected with stable co-lentiviral vectors HA-ERK2 and His-Ubiquitin (Ub) and immunoprecipitated with HA antibody and Protein A/G PLUS-Agarose, then immunoblotted with antibodies against HA and His. b ATDC5 cells were infected with stable co-lentiviral vectors HA-ERK2, His-Ubiquitin (Ub), Flag-USP15, and Flag-USP15C269S. They were immunoprecipitated with HA antibody and Protein A/G PLUS-Agarose, then immunoblotted with antibodies against HA and His. c ATDC5 cells were infected with stable co-lentiviral vectors HA-ERK2, His-Ubiquitin (Ub), Crispr USP15a, and Crispr USP15b. They were co-immunoprecipitated with HA antibody and Protein A/G PLUS-Agarose, then immunoblotted with antibodies against HA and His. d Immunoblot analysis of ATDC5 cells ERK2 overexpression and Crispr USP15b in the presence or absence of MG132 (5 μM) treatment. e ATDC5 cells were infected with stable lentiviral vectors Flag-USP15 and Flag-USP15C269S and treated with cycloheximide (100 μg/mL). Immunoblotting was performed with the indicated antibodies and collected at the indicated times. f The quantification data of ERK protein, Flag, and p-ERK1/2 via Immunoblotting. The data were presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. g Immunofluorescence and DAPI staining of ATDC5 cells were infected with stable lentiviral vectors Flag-USP15 and then detected by USP15 and p-ERK1/2 antibodies (scale bars = 50 μm). h The expression levels of p-ERK1/2 in the two groups were detected by immunohistochemistry (n = 4 for control groups in the OA models, n = 4 for AAV-mediated USP15 overexpression groups in the OA models) (scale bars = 50 μm). i The relative expression of p-ERK1/2 in each group was calculated via immunohistochemistry. The data were presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Data shown are from one representative experiment out of the three performed