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

Figure 3.

Figure 3

Rac1 activation by Hh via Vav2. (A) C3H10T1/2 cells were transiently transfected with the indicated plasmids. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-Smo antibodies. (B) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Vav2 immunoprecipitates (IP, Vav2 Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Vav2 and anti-Smo antibodies. IgG was used as a negative control for IP. (C) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-Vav2 antibodies. IgG was used as a negative control for IP. (D) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-phospho-Vav2 (pVav2) antibodies. IgG was used as a negative control for IP. (E) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 0, 6 or 12 h. (F) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without SAG at 50 nM for 0, 3 or 6 h. (G) Immunofluorescence staining for pVav2 in MEFs with or without N-Shh at 100 ng/ml for 24 h. Primary cilia were indicated by Arl13b staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (H) Hematoxylin-eosin (H&E) staining and immunohistochemistry staining for pVav2 in cerebella slides of GFAP-Cre;SmoM2+/- and SmoM2+/- mice. (I) Immunoblotting analyses of pVav2 and Vav2 in cerebella tissues of GFAP-Cre;SmoM2+/- and SmoM2+/- mice. (J) Immunohistochemistry staining for pVav2 in human clinical sample slides of non-Wnt/Shh-MB and Shh-MB. (K) Rac1 activation assays and immunoblotting analyses for Gli1, pPAK1 as well as PAK1 in C3H10T1/2 cells transfected with or without Myc-Vav2 for 24 h. (L) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 and cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (M) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 shRNA and cultured with or without N-Shh at 100 ng/ml. Total cell lysates were subjected to luciferase assay. N=6. Protein abundance normalized to GAPDH, respectively. *p < 0.05; **, ##p < 0.01; error bar, SD.