Mouse anti centrin

Xenopus embryos were homogenized in lysis buffer [20 mM Tris–HCl: pH 8.0, 5 mM MgCl₂, 1 mM EDTA, 50 mM KCl, 10% glycerol, 0.5% Triton-X, Protease Inhibitor Cocktail (Roche Applied Science)], and embryonic protein extracts were used for immunoblotting with rabbit anti-γ-tubulin (1:1000, abcam), mouse anti-centrin (1:1000, Millipore) and rabbit anti-β-actin (1:2000, Thermo Scientific) antibodies.

Immunoprecipitation and immunoblotting were performed as described previously 6, 20. In brief, transfected cells were solubilized in ice‐cold IP buffer [20 mm Tris–HCl, pH 7.4, 150 mm NaCl, 10 mm Na₂HPO₄, 1% Triton X‐100, 0.5% Na‐deoxycholate, 0.1% SDS, 1 mm EDTA, and 1 mm phenylmethylsulfonyl fluoride (PMSF)]. Where indicated, 2 mm EGTA or 2 mm CaCl₂ was added in lieu of EDTA. Solubilized lysates were incubated for 16 h at 4 °C with protein A and G sepharose beads (GE Healthcare Biosciences, Marlborough, MA, USA) precoated with the indicated rabbit and mouse antibodies, respectively. Protein samples were separated on 7.5–15% SDS/PAGE, transferred to nitrocellulose membranes, followed by immunoblotting. For detecting centrin signal, membranes were fixed with 0.2% glutaraldehyde prior to primary antibody incubation. Input represents 5% of the total protein used for immunoprecipitation. The antibodies include mouse anti‐β‐actin (Sigma, St. Louis, MO, USA), mouse anti‐centrin (Millipore, Billerica, MA , USA), rabbit anti‐rEag1 (Alomone, Jerusalem, Israel), rabbit anti‐GFP (Abcam, Eugene, OR, USA), mouse anti‐GST (Sigma), mouse IgG (Sigma), mouse anti‐Myc (clone 9E10), mouse anti‐PSD‐95 (NeuroMab, Davis, CA, USA), and mouse anti‐synaptophysin. Results shown are representative of at least three independent experiments.