Figure 2.

RPGRIP1L deficiency leads to upregulated MTORC1 signaling. (A) Western blot studies for phospho-(Ser2448)-MTOR in MEFs (n = 3). The amount of phospho-(Ser2448)-MTOR is increased in total cell lysates of Rpgrip1l-negative MEFs. Nonphosphorylated MTOR was used for normalization, while ACT served as a loading control. (B) Immunofluorescence-based measurement of cell size of wild-type and rpgrip1l^−/− limb bud cells. The cells were visualized with TRITC-Phalloidin marking F-ACT to monitor the cell size (in both cases: n = 4). For better visualization the same cells were depicted without and with cell size indication (yellow dotted lines). The cell size of rpgrip1l^−/− limb bud cells is significantly increased. (C) Semiquantitative PCR analysis for Hif1a, a target of MTORC1 signaling (n = 3). The expression of Hif1a is almost doubled in Rpgrip1l-deficient MEFs. For better visualization of the DNA bands, the gel electrophoresis photo was color inverted. (D) Immunofluorescence-based measurement of the OFD1 amount at the base of wild-type and Rpgrip1l-negative MEFs treated with either DMSO (control) or rapamycin. The axoneme was stained with Ac-TUBA (green), the basal body with TUBG (blue), and OFD1 was stained in red (n = 4). Scale bar: 1 µm. Rapamycin treatment resulted in a significant reduction of the OFD1 amount at the ciliary base in both genotypes. (A to D) Asterisks indicate statistically significant differences. (D) The most important significant differences are written in bold.