Anti gli3

The antibodies used in this study included anti-acetylated tubulin (Sigma-Aldrich, T7451 and Cell Signaling Technology, 5335); anti-ARL13B (Proteintech, 17711-1-AP); anti-gamma tubulin (Abcam, ab179503); anti-INPP5E (Proteintech, 17797-1-AP); anti-OSBPL2 (Proteintech, 14751-1-AP and Abclonal, A14199); anti-FLAG (Sigma-Aldrich, F1804); anti-HA (Cell Signaling Technology, 3724); anti-GAPDH (Cell Signaling Technology, 5174); anti–PI(4,5)P₂ (Echelon, Z-P045); anti–PI4P (Echelon, Z-P004); anti-SMO (Santa Cruz, sc-166685); anti-Gli3 (Abcam, ab6050 and Proteintech, 19949-1-AP); anti-Gli1 (Proteintech, 66905-1-Ig); Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (Invitrogen, A31570); Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 (Invitrogen, A10040); Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Invitrogen, A21202); Donkey F(ab′)2 Anti-Rabbit IgG H&L, Alexa Fluor 647 (Abcam, ab181347); IRDye 800CW Secondary Antibody (LI-COR, 925-32211); and IRDye 680LT Secondary Antibody (LI-COR, 925-68020). The regents used in this study included Smoothened Agonist (Sigma-Aldrich, 566661); Digitonin (MCE, HY-N4000); FLAG Immunoprecipitation Kit (Millipore, FLAGIPT1); DAPI (Sigma-Aldrich, F6057); and isopropyl β-D-thiogalactoside (IPTG, Sigma-Aldrich, I6758).

Western blotting was performed as described previously^{27 (link)}. Briefly, tissue or cell samples were lysed on ice in RIPA lysis buffer containing 1% PMSF (Beyotime, China). Total protein samples (40 μg) were separated by SDS-PAGE and then transferred onto a PVDF membrane. Primary antibodies were used as follows: anti-E-cadherin (Cell Signaling, 3195), anti-VEGF-A (Abcam, ab46154), anti-Sonic Hedgehog (Abcam, ab53281), anti-Gli2 (Abcam, ab2605556), anti-Gli3 (Abcam, ab6050), anti-p-AKT T308 (Cell Signaling, 4056), anti-p-AKT S473 (Cell Signaling, 4046), anti-pan-AKT (Cell Signaling, 4691), Beclin1 (Cell Signaling, 3495), and anti-LC3B (Sigma-Aldrich, L7543). GAPDH (Proteintech, 600004-1-Ig) or β-actin (Cell Signaling, 4970) was used as an internal standard. Immunofluorescent anti-rabbit and anti-mouse secondary antibodies were purchased from LI-COR Bioscience (Lincoln, USA), and the signals were visualized with an Odyssey Infrared Imaging System (Lincoln, USA). Anti-rabbit-HRP and anti-mouse-HRP secondary antibodies were purchased from Beyotime (Shanghai, China). After ECL exposure, images were captured by an Amersham Imager 600 (GE, USA). ImageJ software was used to quantify the immunoreactive bands.