MKRN1 Ubiquitylates p21 to Protect against Intermittent Hypoxia-Induced Myocardial Apoptosis

Figure 6

Under IH conditions, MKRN1 induced p21 ubiquitination and proteasome pathway degradation. (a) H9C2 cells were subjected to IH stimulation. Before harvest, cells were treated with DMSO, 20 μM MG132 ((a), 1), or 25 mmol/L NH₄Cl ((a), 2) for 3 h and representative immunoblotting analysis and quantification of p21 (), GAPDH as an internal control; ; (b) H9C2 cells were transfected with MKRN1-overexpressing lentiviruses or vector; before harvest, cells were treated with or without 20 μM MG132 for 3 h and representative immunoblotting analysis and quantification of p21 () and GAPDH as an internal control; versus the blank group, versus the MG132 group, versus the MKRN1 group; (c) H9C2 cells were subjected to IH stimulation. Cell lysate and normal rabbit IgG antibody or p21 antibody were subjected to CO-IP, and the indicated antibodies were used for WB analysis; (d) 293T cells were transfected with plasmids expressing Flag-MKRN1, Myc-p21, and His-Ub before IH stimulation. Before harvest, cells were treated with MG132 for 3 h, the Myc antibody was used to purify Myc-p21 using CO-IP, and the His antibody was used for WB analysis; (e) cells in Figure 6(d) were subjected to NC or IH stimulation; before harvest, cells were treated with MG132 for 3 h, the Myc antibody was used for CO-IP, and the His antibody was used for WB analysis; (f) H9C2 cells transfected with siControl or siMKRN1-2 were subjected to IH stimulation, and MG132 was added 6 h prior to harvest. The whole-cell lysate was subjected to CO-IP using the p21 antibody, and the Ub antibody was used for WB analysis (the second and third columns), with normal rabbit IgG as the negative control (the first column). indicates the independent sample number in each group.

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