Mouse anti γ tubulin

Primary antibodies used for immunocytochemistry (ICC) or immunohistochemistry (IHC) included mouse anti-acetylated alpha-tubulin (1:3000 (ICC/IHC); Sigma (cat # T6793; lot # 088K4829)), rabbit anti-Arl13b (1:3000 (IHC); Proteintech (cat # 17711-1-AP; lot # 00017960)), mouse anti-gamma-tubulin (1:2000 (ICC/IHC); Sigma (cat # T6557; lot # 072M4808)), rabbit anti-Gli3C (1:1000 (ICC); gift from S. Scales) [35 (link)], rabbit anti-PCM1 (1:1000 (ICC/IHC); Bethyl Laboratories (cat # A301-150A; lot # A301-150A-1)), rabbit anti-SMO (1:1000 (ICC/IHC); Abcam (cat # ab38686; lot # GR198520)). Cells and sections stained sequentially with mouse antibodies against gamma- and acetylated alpha-tubulin were blocked with anti-mouse Fab fragments (20 μg/ml; Jackson Immunoresearch (cat # 715-007-003; lot # 114326)) as previously described [24 (link)]. Secondary antibodies were species-specific and were conjugated with fluorescent tags (1:400 (ICC/IHC); Jackson Immunoresearch). Stained sections and cells were coverslipped with Prolong Gold antifade media containing DAPI (Invitrogen).

Harvested cells were lysed by vortexing vigorously with glass beads in HB buffer (25 mM MOPS (pH7.2), 5 mM ethylene glycol bis-(2-aminoehylether) tetraacetic acid (EGTA) (pH7.2), 15 mM MgCl₂, 150 mM KCl, 50 mM beta-glycerophosphate, 15 mM p-nitrophenylphosphate, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.1 mM Na₃VO₄, 0.2% NP-40, protease inhibitor cocktail (Complete Mini EDTA-free, Roche)). The following antibodies were used to detect target proteins: rabbit anti-HA Y11 (Santa Cruz, Dallas, Texas), mouse anti-GFP (Roche), rabbit anti-GFP (Life Technologies, Carlsbad, California), mouse anti-γ-tubulin (Sigma), rabbit peroxidase anti-peroxidase soluble complex antibody (Sigma, St. Louis, Missouri), and mouse anti-Myc 9E10 (Santa Cruz).

The following primary antibodies were used: mouse anti–γ-tubulin (1:500; Sigma-Aldrich, St. Louis, MO, United States), mouse anti–α-tubulin (1:500; Santa Cruz Biotechnology, Dallsa, TX, United States), mouse anti-GFP (1:500; Roche, Basel, Switzerland), rabbit anti-GFP (1:500; Abcam, Cambridge, United Kingdom), mouse 22C10 (1:200; DSHB), mouse 21A6 (1:200; DSHB), iFluor^TM 555 Phalloidin (1:200; Yeasen, Shanghai, China). The following secondary antibodies were used: goat anti–mouse Alexa Fluor 488 or Alexa Fluor 594 or goat anti–rabbit Alexa Fluor 488 or Alexa Fluor 594.

Testes were dissected in 1× PBS and fixed in 4% formaldehyde for 30 min. For immunostaining, testes were incubated with primary antibodies overnight at 4°C, followed by washes in 1× PBST and incubation with secondary antibodies for two hours at RT. The following primary antibodies were used: rabbit anti-Lid (1:1000; from Julie Secombe, Albert Einstein College of Medicine, Bronx, NY, USA); mouse anti-Armadillo [1:100; developed by Eric Wieschaus, Princeton University, Princeton, NJ, USA, and obtained from Developmental Studies Hybridoma Bank (DSHB)]; rat anti-Vasa (1:100; developed by Allan Spradling and Dianne Williams and obtained from DSHB); rabbit anti-Vasa (1:100; Santa Cruz, sc-30210); chicken anti-GFP (1:1000; Abcam, #13970); rabbit anti-Stat92E (1:800; from Denise Montell, Johns Hopkins School of Medicine, Baltimore, MD, USA); rabbit anti-phospho-Histone H3 (Thr3) (1:200; Millipore, #05-746R); mouse anti-α-spectrin (1:50; obtained from DSHB); mouse anti-γ-tubulin (1:100, Sigma, GTU-88); mouse anti-FasIII (1:50; obtained from DSHB, 7G10); rabbit H3K4me3 (1:200; Cell Signaling, #9751S); rabbit anti-Zfh1 (1:5000; from Ruth Lehmann, Skirball Institute of Biomolecular Medicine, NY, USA). Alexa Fluor 488, 568 and 633-conjugated Goat anti-mouse, anti-rabbit, and anti-rat secondary antibodies were used (1:200; Molecular Probes/Invitrogen).

The following primary antibodies diluted in PBST were used in these experiments: mouse anti-Delta, mouse anti-Arm (Developmental Studies Hybridoma Bank, Iowa City, IA, USA), 1:200; mouse anti-GFP and rabbit anti-GFP (Molecular Probes, Eugene, OR, USA), 1:1000; rat anti-GFP (Nacalai Tesque Inc., Kyoto. Japan), 1:1000; rabbit anti-γH2AvD (Rockland, Gilbertsville, PA, USA) 1:2000; rabbit anti-pS/TQ (Cell Signaling Technologies, Danvers, MA, USA), 1:1000; rabbit anti-phospho-histone H3 (PH3, Millipore, Billerica, MA, USA), 1:1000; mouse anti-γ-tubulin (Sigma-Aldrich), 1:1000; rabbit anti-β-gal (Upstate Biotechnology Inc., Lake Placid, NY, USA), 1:1000; and anti-CCleaved caspase-3 (Cell Signaling Technologies), 1:1000; rabbit anti-pJNK antibody (Cell Signaling Technologies). The following secondary antibodies diluted in PBST were used: goat anti-rabbit FITC (Jackson ImmunoResearch, West Grove, PA, USA), 1:400; goat anti-rabbit Cy3 (Jackson ImmunoResearch), 1:400; goat anti-mouse FITC (Jackson ImmunoResearch), 1:400; goat anti-mouse Cy3 (Jackson ImmunoResearch), 1:400; goat anti-rat FITC (Jackson ImmunoResearch), 1:400, goat anti-rabbit Alexa Fluor^® 647 (Jackson ImmunoResearch), 4′,6-diamidino-2-phenylindole (DAPI, Molecular Probes), 1:1000.