Mouse anti akap4

Testis and epididymides were dissected from wild-type and Tcp11^−/− adults and placed into PBS. Testis samples were lysed in 1 mL RIPA (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM DTT, 1% Nonident P-40, 0.5% deoxycholate, 0.1% SDS, protease inhibitors) using Qiagen’s TissueLyser II (30 Hz, 2 min, RT). Epididymides were first minced with dissecting scissors in 1 mL RIPA (50 mM Tris pH 7.5, 150 mM NaCl, 5 mM DTT, 1% Nonident P-40, 0.5% deoxycholate, 0.1% SDS, protease inhibitors) and then lysed with Qiagen’s TissueLyser II (30 Hz, 2 min, RT). Lysates were clarified with a 10 min spin at 18 000×g at 4 °C. The protein concentration was determined with the Bradford method. Thirty microgram per sample were loaded per well. Western blot analysis was performed using BioRad’s TransBlot Turbo. PVDF membranes were blocked with TBS with 0.05% Tween and 5% milk and washed with TBS with 0.05% Tween and 0.5% milk. Both primary and secondary antibodies were diluted in washing buffer. Nacalai’s Super Signal was used for chemiluminescence and detected with Image Quant. Antibodies used include the following: goat anti-BASAGIN 1:1000 (Santa Cruz), mouse anti-AKAP4 1:1000 (BD Biosciences), and mouse anti-Acetylated Tubulin 11 000 (Sigma).

Immunoblot analysis was performed as described previously.12 Samples were subjected to SDS‐PAGE followed by western blotting. After blocking with 10% skim milk, blots were incubated with primary antibodies overnight at 4°C and then incubated with secondary antibodies conjugated to horseradish peroxidase (1:10,000, #805‐035‐180, #111‐036‐045, #115‐036‐062, or #112‐035‐167, Jackson ImmunoResearch, PA, USA) for 2 hours at room temperature. Antibodies used: goat anti‐KIF9 1:100 (#SC99958, Santa Cruz Biotechnology, CA, USA); rabbit anti‐ACTB 1:1000 (#PM053, Medical & Biological Laboratories, Aichi, Japan); goat anti‐BASIGIN 1:500 (#SC9757, Santa Cruz Biotechnology), mouse anti‐acetylated tubulin 1:1000 (#T7451, Sigma‐Aldrich, MO, USA); mouse anti‐AKAP4 1:5000 (#611564, BD Biosciences, CA, USA); mouse anti‐phosphotyrosine 1:1000 (#05‐321, Merck Millipore, MA, USA); rabbit anti‐RSPH9 1:200 (#HPA031703, Atlas Antibodies); rat anti‐PA 1:1000 (#012‐25863, FUJIFILM Wako Pure Chemical, Osaka, Japan); and rabbit anti‐FLAG 1:1000 (#PM020, Medical & Biological Laboratories). Immunoreactive proteins were detected by an ECL western blotting detection kit (GE Healthcare, Little Chalfont, UK).