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. 2023 Nov 13;223(1):e202203005. doi: 10.1083/jcb.202203005

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© 2023 Wu et al.

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Figure S3. — Related to Fig. 6. CDK2 regulates USP37 stability by phosphorylating and activating USP37. (A) Cells were treated with/without JNJ-7706621 for the three different point times (0, 6, and 12 h) before harvest, and then cell lysates were blotted with the indicated antibodies. (B) Cells stably expressing control shRNA or USP37 shRNA were treated with/without JNJ-7706621 before harvest, and then cell lysates were blotted with the indicated antibodies. (C) Immunoreactive bands of at least three independent experiments were quantified using ImageJ software. Relative ERK1/2 and USP37 protein levels were quantified with GAPDH from B as internal standards. Data were represented as means ± SD. (D) Cells transfected with the indicated constructs were treated with/without JNJ-7706621, and then treated with MG132 for 10 h before harvested. Covalently modified proteins purified on NiNTA-agarose under denatured conditions. Ubiquitinated ERK1/2 was detected by anti-Myc antibody. (E) Cells transfected with the indicated constructs were with/without NJ-7706621 and meanwhile treated with MG132 for 10 h before being harvested. Covalently modified proteins were purified on NiNTA-agarose under denatured conditions. Ubiquitinated USP37 was detected by anti-HA antibody. (F) Cells stably expressing control or CDK2shRNAs were transfected with His-ub, and then were treated with MG132 for 12 h before harvested. Covalently modified proteins purified on NiNTA-agarose under denatured conditions. Ubiquitinated USP37 was detected by anti-HA antibody. (G) Cells expressing Control, CDK2shRNA1, and CDK2shRNA2 were treated with cycloheximide (CHX) and then harvested at the indicated four-point times (0, 3, 6, and 10 h). The samples were immunoblotted with indicated antibodies. (H) Quantification of the USP37 protein levels relative to GAPDH shown in G using ImageJ software. (I) HEK293T cells were transfected with Myc-ERK1 plasmid together with His-ub, and then treated with as indicated. Cell lysates were purified with anti-Myc beads and immunoblotted with indicated antibodies. (J) Cells were transfected with the indicated constructs and treated with JNJ-7706621 before harvest, and cell lysates were blotted with the indicated antibodies. Source data are available for this figure: SourceData FS3.