Figure 7

RGS7 is degraded by the proteasome in Ate1-dependent manner. (A) Western blot of RGS7 exogenously expressed in wild type (WT) and Ate1 knockout (KO) mouse embryonic fibroblasts transfected with Rgs7. 12 hours after transfection (lane 2), cells were further incubated with the proteasome inhibitor MG132 and were harvested at the indicated time points (lane 3 to 6). RGS7 protein level increases over time only in WT in the presence of MG132. Lane 1 in each panel shows the blot of untransfected control cells. (B) Western blot quantification of RGS7 in cells harvested after 2 hours of incubation with MG132, RGS7 without MG132 incubation, and RGS7 in untransfected control. RGS7 protein level significantly increased with MG132 treatment in WT, but not in KO cells. Data points from 4 independent experiments are plotted with a unique symbol for each experiment. (C) Western blot quantification of RGS9 in WT and KO cells from 3 independent experiments. The effect of MG132 treatment on RGS9 protein levels does not depend on the WT or KO background, indicating that its degradation is not affected by Ate1. In all colored panels of (B and C), RGS protein levels were first normalized to GAPDH loading control and then normalized for each blot. The error bars represent SEM. The p-values are from paired Student’s t-test. (D) The ratio of RGS7 level without/with MG132 was significantly higher in KO than WT cells while the ratio of RGS9 level without/with MG132 in KO and WT cells did not differ significantly. The error bars represent SEM. The p-values are from Welch’s t-test. (E) Western blot quantification of the stable RGS levels after 12 hours of transfection with GAPDH as loading control. Data from each experiment are plotted with a unique symbol. The error bars represent SEM. However, these cannot be compared directly due to the unknown factors of transfection efficiencies and GAPDH levels potentially different between WT and KO as well as antibody differences. The meaningful comparison is to compare the ratio of two RGS intensities between KO and WT, such that the factors cancel out—assuming they are constant and multiplicative. The ratios are shown in (F). Calculation of each average ratio was performed in log2 unit (see "Methods") and plotted in linear fashion, normalized to WT. The error bars represent STD. The p-values are from Welch’s t-test.