Anti gamma tubulin

Transduced HeLa, NHEK, and serum-starved hTERT-RPE1 cells were washed with PBS and harvested in the FLAG lysis buffer. Supernatants were incubated with 30 μL anti-FLAG-beads (ANTI-FLAG^TM M2 Affinity Gel) overnight at 4 °C and 1 h at RT on a rotator to pool-down ARP-T1-FLAG proteins. Beads were separated by centrifugation for 3 min at 5000 × g, washed five times with TBS, and eluted in 23 μL TBS and 7 μL 5x SDS-sample buffer at 85 °C for 5 min. ARP-T1 precipitation was confirmed using ARP-T1 antiserum (1:2000, GP-SH6), and co-precipitated proteins were analyzed using anti-acetylated tubulin (1:1000, T6793, Sigma-Aldrich), anti-TCP8 / TCP1 theta (1:500, PA5-30403, Thermo Scientific), anti-HSC70 (1:200, PA5-27337, Thermo Scientific), anti-BAG2 (1:100, PA5-30922, Thermo Scientific), anti-gamma tubulin (1:500, ab11316, Abcam), anti-EDH4 (1:1000, Dr. Plomann’s lab), anti-septin 2 (1:2000, HPA018481, Sigma-Aldrich), anti-septin 9 (1:2000, HPA042564, Sigma-Aldrich).

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).

Oocytes were exposed to acidic tyrode solution (pH 2.5) for a few seconds to remove the zona pellucida followed by three washes in M2 medium. Oocytes were then fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) in phosphate-buffered saline (PBS) at room temperature for 30 min, followed by permeabilization in PBS containing 0.5% Triton X-100 for 2 h at room temperature. Sample blocking was conducted with 1% bovine serum albumin (BSA, Amresco, Solon, OH, USA) in PBS containing 1/1000 Tween-20 (Amresco) and 1/10,000 Triton X-100. After blocking, samples were incubated with primary antibodies overnight at 4°C. For primary antibodies, we used mouse anti-alpha tubulin (1:2000, Abcam, Cambridge, UK) and anti-gamma tubulin (1:500, Abcam). For secondary antibodies, we used DyLight 549-conjugated donkey anti-mouse (1:100, Jackson ImmunoResearch Laboratories, West Grove, PA, USA). DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI, 5 μg/mL, Roche, Mannheim, Germany) for 10 min. After staining and washing, samples were mounted on glass slides using Vectashield mounting medium (Vector Labs, Burlingame, CA, USA) and examined with a confocal laser-scanning microscope (Nikon, A1R, Tokyo, Japan). Images were analyzed with NIS-Element AR 3.0 software. Chromosome spreading analysis was performed as we described previously [63 (link)].