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. 2023 Jan 26;34(4):590–606. doi: 10.1681/ASN.0000000000000082

Figure 7.

Figure 7

Kinase activity is dispensable for mTOR pathway activity. (A) Western blot showing that human GRK4 variants are expressed in GRK4 knockout cells after transient transfection. Empty vector (EV) or plasmids encoding human GRK4 variants were transiently transfected. n=2. (B) Expression of human kinase-dead GRK4 (K216,217M) significantly reduces cell size similar to WT GRK4, while patient-derived variants do not. n=4 experiments with 641 (EV), 820 (WT), 776 (K215,216M), 739 (GRK4 R65L), and 675 (GRK4 A142V) cells. Kruskal-Wallis test with Dunn multiple comparisons test. ns: P=0.1299, *P=0.0140, and ****P< 0.0001. (C) Protein content in GRK4 KO cells is increased but can be normalized by rapamycin treatment. n=4 experiments. One-way ANOVA with Sidak multiple comparisons test. **P=0.0070 and *P=0.0130. (D) Protein content in GRK4 KO cells can be rescued by kinase-dead GRK4, but not by patient variants. n=4 experiments. One-way ANOVA with Holm-Sidak multiple comparison test. ns: P=0.8687, *P=0.0241 (EV versus GRK4 WT), 0.0169 (EV versus GRK4 R65L), and (0.0276 EV versus GRK4 A142V). EV, empty vector.