The ERAD pathway also contributes to ROMK mutant turnover in HEK293 cells.

(A) Stability assays of HEK293 cells expressing wild-type ROMK, or ROMK carrying the G228E, L320P, or N377K mutation. HEK293 cells transfected with the indicated expression vector were treated with MG-132 or the equivalent volume of DMSO for 30 min, at which point cycloheximide was added. Cells were next processed as described in the Materials and Methods. Representative immunoblots are shown, and graphs show the percentage of the protein remaining over time, compared to the 0 hr (h) time point. A rabbit antiserum was used to detect ROMK [160], and a mouse monoclonal antibody against actin was used as a loading control. All bands present in the ROMK immunoblots are specific for the protein and were used for the quantification. (B) Steady-state protein levels before and after p97 inhibition with CB-5083 for 4 hrs. Graphs were made using GraphPad Prism (ver. 9.5.0), and data represent the means of at least three independent experiments, ± S.E. (error bars). For each experiment, a representative immunoblot is shown, and the quantification was performed using ImageJ (ver. 1.53c). Since the top-most band in the ROMK immunoblots represents a non-specific protein species recognized by the ROMK antiserum, the bands in the center of the blots were used for the quantification. p-values in were calculated with two-tailed Student’s t-test for independent samples. ns, p ≥ 0.05; *, p < 0.05.