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

Figure 5.

Figure 5

Phosphorylated-KIF3A by Rac1 activation binds to and stabilizes IFT88 protein. (A) C3H10T1/2 cells were transfected with or without daRac1 and cultured with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (B) C3H10T1/2 cells were cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (C) Immunoblotting analyses of IFT88 in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and transfected with or without KIF3A for 24 h and treated for different time periods with CHX. (D) Immunoblotting analyses of phospho-KIF3A (pKIF3A, pSer/Thr) and KIF3A in C3H10T1/2 cells transfected with or without daRac1 for 24 h. (E) Immunoblotting analyses of pKIF3A and KIF3A in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h. (F) Conserved phosphorylation sites in KIF3A by PAK1 among species. (G) C3H10T1/2 cells were transfected with or without wild-type Flag-KIF3A (WT) or Flag-KIF3A mutant (S689A, T694A, S698A, 3Mut) and cultured for 48 h. Total cell lysates (Input) and anti-Flag immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Flag and anti-PAK1 antibodies. (H) C3H10T1/2 cells were transfected with the indicated plasmids and cultured with NSC23766 at 10 μg/ml for 48 h. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-HA antibodies. (I) An in vitro kinase assay of GST-KIF3A and PAK1* in the presence or absence of ATP. Phospho-Ser/Thr (pS/T) was analysed by western blot. (J) Immunofluorescence staining for IFT88 in Kif3a-knockout C3H10T1/2 cells transfected with KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with NSC23766 at 10 μg/ml for 48 h. Primary cilia were indicated by Ac-Tub staining. Bar, 50 μm. (K) Kif3a-knockout C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with wild type KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. Total cell lysates were subjected to luciferase assay. N=6. (L) Kif3a-knockout C3H10T1/2 cells were transiently transfected with KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. mRNA levels of Gli1 and Ptch1 were analyzed. N=6. RNA and protein abundance normalized to GAPDH, respectively. **p < 0.01; error bar, SD.