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. 2011 Jun 8;286(31):27676–27686. doi: 10.1074/jbc.M111.234666

FIGURE 1.

FIGURE 1.

Gas8 is important for Smo signaling and cilary localization. A and B, association between Smo and Gas8 was assayed in vitro by yeast two-hybrid technique using the last 211 residues of human Smo as a bait (SmoCT) and Gas8 cDNA as prey, either encoding Gas8 residues 357–478 (Gas8, A) or its 93 C-terminal residues (Gas8CT, B) under screening conditions lacking histidine and adenine (-HIS, -ADE). C, Smo and Gas8 specifically interact in HEK cells. HEK cells were transiently transfected with pcDNA3.1 control vector, Myc-tagged Smo, Flag-tagged mouse Gas8, or both Myc-Smo and Gas8. Lysates were immunoprecipitated (IP) with an antibody directed to the Flag tag and immunoblotted for the presence of Myc-Smo or the Flag-tag, WCL, whole cell lysate. D–K, lentiviral shRNA-mediated Gas8 knock down. D, Gas8 protein levels were detected in lysates from NIH-3T3 cells stable for either Gas8 shRNA or a scramble control (scr) shRNA. Anti-actin antibody was used for loading control. Averages ± S.E. were calculated from 4 experiments and presented as percent of scr control. E, shRNA stable cell lines were transiently transfected with a Gli luciferase reporter and Renilla luciferase internal control. Cells were treated with either SAG or DMSO for 24 h and then starved overnight in the induction media and lysed. Averaged fold induction in luciferase activity values ± S.E. (SAG/DMSO) were calculated. *, p < 0.05. F, GFP-Smo cell line was infected with the lentiviral shRNAs to generate double stable lines, plated, and stained for acetylated tubulin (red). G–I, percent of cells with cilia (G), the averaged length of the cilia (H), and the percent of GFP-Smo containing cilia (I) ± S.E. were calculated from four experiments in GFP-Smo cells stable for either Gas8 or scr shRNAs. **, p < 0.01. J, GFP-Smo protein levels were detected in the double stable cell lines using an antibody against GFP. K, Gas8 protein levels were detected upon scr shRNA following stimulation with SAG or DMSO. In both J and K, anti-actin antibody was used for loading control. All p values were calculated by two-tailed Student's t test.