T396I Mutation of Mouse Sufu Reduces the Stability and Activity of Gli3 Repressor
Fig 2
SufuT396I does not stabilize Gli3FL protein and reduces the processing of Gli3FL.
(A) Western blotting of lysates prepared from SufuR146X/R146X at E9.5 and SufuT396I/T396I, SufuT396I/+, and wild-type embryos at E10.5 with anti-Gli3, anti-Sufu, and anti-actin antibodies. Each image presented in the Fig. is a representative of independent triplicated experiments. The full gel images are shown in S6A Fig. (B, C) Relative expression of Gli3FL (B) and Gli3REP (B and C). Western blotting was performed two times using lysates prepared from five wild-type and five SufuT396I/T396I embryos at E10.5 (S2A Fig.). Expression levels were quantified from the band intensity shown in S2A Fig. as relative values of the Gli3FL/actin and Gli3REP/actin expression ratios (B) and the direct ratio of Gli3REP/Gli3FL (C). (**) p < 0.01, two-tailed Student’s t-test. Error bars indicate the standard deviations. (D) Western blotting of cell lysates from wild-type and SufuT396I/T396I MEFs with indicated antibodies. The SufuT396I/T396I MEFs were electroporated with 10.0, 1.0, or 0.1 μg of the HA–Sufu construct (lane 3, 4, and 5, respectively), or 10 μg of the HA–SufuT396I construct (lane 6). The mobilities on SDS-PAGE of the wild-type Sufu (lane 3–5) and SufuT396I (lane 6) are identical. The complete gel images are shown in S6B Fig. This image is representative of two independent experiments. (E) Western blotting of cell lysates from Sufu−/− cells with indicated antibodies. The Sufu−/− cells were electroporated with a mixture of the Flag–Gli3 construct (6 μg) and the HA–Sufu construct (4.00, 1.33, 0.44, or 0.15 μg) for the wild-type Sufu cotransfection (lane 2 to 5, respectively), or a mixture of the Flag–Gli3 construct (6 μg) and the HA–SufuT396I construct (4 μg) for mutant Sufu cotransfection (lane 6). The complete gel images are shown in S6C Fig. This image is representative of two independent experiments. (F) Western blotting of immunoprecipitates or lysates from 293T cells transfected with expression constructs as indicated at the top. The complete gel images are shown in S6D Fig. This image is representative of two independent experiments.