Skip to main content
. 2022 Jan 1;12(3):1303–1320. doi: 10.7150/thno.67702

Figure 2.

Figure 2

Rac1 activation by Hh and regulation of Rac1-mediated Hh via PAK1. (A) Immunofluorescence staining for Smo in MEFs cultured with or without N-Shh at 100 ng/ml for 48 h and NSC23766 (NSC) at 10 μg/ml for 24 h. Primary cilia were indicated by Ac-Tub staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (B,C) Rac1 activation assays in C3H10T1/2 cells transfected with caSmo for 24 h (B) or cultured with SAG at 50 nM for 24 h (C). (D) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (B) and (C). (E,F) Rac1 activation assays in C3H10T1/2 cells transfected with siSmo (E) for 72 h or cultured with Cyclopamine (Cyc., F) at 5 μM for 24 h. (G) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (E) and (F). (H) Immunoblotting analyses of phospho-PAK1 (pPAK1) and PAK1 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 24 h. (I) Immunoblotting analyses of pPAK1 and PAK1 in cerebellum tissues of GFAP-Cre;SmoM2+/- and SmoM2+/- mice. (J) Quantification via densitometry (n=3) and statistical analysis of pPAK1 bands of (H) and (I). (K) Immunoblotting analyses of pPAK1 and PAK1 in C3H10T1/2 cells transfected with or without daRac1 for 24 h and cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (L) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs from Shh+/- and Shh-/- embryos. (M) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (N) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with the daRac1 and cultured with or without N-Shh at 100 ng/ml or IPA-3 at 1 μM for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (O) C3H10T1/2 cells were cultured with or without IPA-3 at 1 μM for 24 h. Total cell lysates (Input) and anti-SuFu immunoprecipitates (IP, SuFu Ab, +) from total cell lysates were analyzed by immunoblotting with anti-SuFu and anti-Gli1 antibodies. Protein abundance normalized to GAPDH, respectively. **, ##p < 0.01; error bar, SD.