Anti phosphotyrosine antibody

PC-3M-luc-C6 cells were serum-starved for 1 hour in serum-free medium and treated for 1 hour with 0.2 μg/mL human Fc (as a negative control), 0.2 μg/mL ephrin-A1 Fc (as a positive control), 100 μM YSA-L2-PTX or dYNH-L2-PTX or DYP-L2-PTX. The cells were then placed on ice, rinsed once with cold PBS and incubated for 20 min at 4°C with a 0.5 mg/mL EZ-link sulfo-NHS-biotin (Thermo Scientific/Pierce, Rockford, IL) in PBS. The cells were then washed 3 times with a 100 mM glycine in PBS to quench the biotinylation reaction, followed by PBS. The cells were lysed in modified RIPA lysis buffer (150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 20 mM Tris, pH 8.0) containing protease inhibitors and 1 mM sodium orthovanadate. For immunoprecipitations, the lysates were incubated with 1 μg anti-EphA2 antibody (Millipore-Upstate, Inc. Temecula, CA) immobilized on GammaBind Sepharose beads (GE Healthcare Life Sciences). Immunoprecipitates and lysates were probed by immunoblotting with an anti-phosphotyrosine antibody (Millipore, Inc, Temecula, CA), streptavidin coupled to HRP (Thermo Scientific/Pierce, Rockford, IL), or anti-EphA2 antibody (Life Technologies/Invitrogen). Lysates of PC-3M-luc-C6 cells were probed by immunoblotting with the EphA2 Millipore antibody and with a GAPDH antibody (AbCam).

Cultured adrenal glomerulosa cells on Falcon Primaria dishes were incubated with eqKRB+ in the presence or absence of AngII for 30 min. The supernatants were collected for aldosterone assay, and the cells were washed at least three times with ice cold 0.9% NaCl, after which the cells were collected on ice in 500 μl of IP lysis buffer. The IP lysis buffer was composed of the following: 50 mM Tris (pH 7.5), 150 mM NaCl, 1% NP-40, 1 mM MgCl₂, 5 mM NaF, 1 mM Na₃VO₃, and 1 Complete Protease Inhibitor Cocktail Tablet (Roche Pharmaceuticals, San Francisco, CA) per 7 mLs of buffer. The cells were incubated with either anti-PKCμ/PKD (Santa Cruz) or anti-phosphotyrosine antibody (Millipore) for 2 hours at 4°C, after which the samples were incubated with Tru-blot anti-rabbit IgG beads (eBioscience) for 1 hour. The samples were then subjected to SDS-PAGE as described above.