Rabbit anti apkc c 20

Embryos were dechorionated in 2% sodium hypochlorite for 5 min and heat fixed in 5 ml of E wash (7% NaCl and 0.5% Triton X-100) at 80°C, which was immediately cooled down by the addition of 15 ml of ice-cold E wash. Embryos were then rinsed with PBS and placed in methanol under a heptane phase, devitelinized by strong agitation, and further incubated for 1 h in fresh methanol. Embryos were saturated in NGT (2% normal goat serum and 0.3% Triton X-100 in PBS). Primary antibodies were diluted in NGT and incubated overnight at 4°C under agitation. Primary antibodies used were mouse anti-Dlg (1:10 dilution; clone 4F3; Developmental Studies Hybridoma Bank), rat anti-Crb (1:500; Pellikka et al., 2002 (link)), guinea pig anti-Yrt (1:250; Laprise et al., 2006 (link)), rabbit anti-aPKC C-20 (1:250; Santa Cruz Biotechnology, Inc.), and mouse anti-Flag (1:250; clone M2; Sigma-Aldrich). Secondary antibodies were conjugated to Cy3 (Jackson ImmunoResearch Laboratories, Inc.), Alexa Fluor 488 (Molecular Probes), or Alexa Fluor 647 (Jackson ImmunoResearch Laboratories, Inc.) and used at a dilution of 1:400 in NGT (1 h at room temperature).

Embryos were dechorionated in 2% sodium hypochlorite for 5 min, rinsed with water, homogenized in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.75% Nonidet P-40, 0.1 mM sodium orthovanadate, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM NaF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 0.7 µg/ml pepstatin), and processed for SDS-PAGE and Western blotting as previously described (Laprise et al., 2002 (link)). Primary antibodies used were guinea pig anti-Yrt (1:5,000; Laprise et al., 2006 (link)), rabbit anti-aPKC C20 (1:2,000; Santa Cruz Biotechnology, Inc.), mouse anti-Flag (1:2,500; clone M2; Sigma-Aldrich), mouse anti-Actin (1:10,000; clone C4; EMD Millipore), guinea pig anti–Par-6 (1:2,000; Kim et al., 2009 (link)), and rabbit anti-GST (1:8,000; provided by J.-Y. Masson, Université Laval, Québec, Québec, Canada). HRP-conjugated secondary antibodies were used at a 1:1,000 dilution. For λ Phosphatase assays, embryos were lysed in lysis buffer without phosphatase inhibitors. λ Phosphatase (New England Biolabs, Inc.) was used following the manufacturer’s recommendations.