Mouse anti na k atpase

Cells were lysed on ice for 20 min with NP-40 buffer containing 10 mM Tris-HCl, pH 7.4, 120 mM NaCl, 1% NP-40, and proteases inhibitors (Roche Applied Science #04693124001). Cell lysates were spun at 10,000 × g for 30 min at 4 °C. Sixty micrograms of proteins were mixed with a 4x Laemmli buffer and then loaded on SDS-PAGE. Proteins resolved onto 4–20% polyacrylamide gels (Bio-rad #4561096) were transferred to Hybond C nitrocellulose membranes (GE Healthcare #10600016). Membranes were immunoblotted with primary antibodies (rabbit anti-GFP, 1:10000, Torrey Pines Biolabs #TP401; rabbit anti-α-actinin, clone D6F6, 1:3000, Cell Signaling #6487; mouse anti-Na⁺/K⁺-ATPase, 1:5000, Abcam #AB7671; rabbit anti-calreticulin, 1:1000, Abcam #92516) and then with either an anti-rabbit or anti-mouse horseradish peroxidase (HRP)–conjugated secondary antibody (anti-rabbit, 1:10000, GE Healthcare #NA934; anti-mouse, 1:5000, Merck #12-349). Immuno-reactivity was detected with an enhanced chemiluminescence substrate for detection of HRP (SuperSignal West Pico Chemiluminescent Substrate, Thermo Fisher Scientific #34080).

A custom rabbit anti-heparanase 2 antibody was designed and manufactured (Generon). The immunizing peptide sequence used was QLDPSIIHDGWLDC (Fig. 1). Commercially available antibodies used in this study were: mouse anti-acetylated tubulin (Sigma), mouse anti-12/101 (Developmental Studies Hybridoma Bank), rabbit anti-ERK (Cell Signalling), mouse anti-pERK (Sigma–Aldrich), mouse anti-Na,K-ATPase (Abcam), rabbit anti-LRIG2 (Abgent), horseradish peroxidase (HRP)-coupled anti-mouse and anti-rabbit (Life Technologies). To test specificity, anti-heparanase 2 antibody was incubated overnight at 4°C on a rotator with a 50-fold excess immunizing peptide or a control reaction of 50-fold excess non-specific peptide, prior to use in IHC applications. Western blotting was performed by standard methods. Total protein was extracted from pools of three embryos, with amounts equivalent to one embryo separated via SDS–PAGE.