Agarose conjugated vdr antibody

Placental cytosol proteins were prepared in a lysis buffer (0.6% Nonidet P-40, 0.5% sodium deoxycholate, 150 mM NaCl and 50 mM Tris-Hcl, pH 7.5) containing 0.1 mM vanadyl sulfate and protease inhibitors (0.5 mg/ml aprotinin, 0.5 mg/ml trans-epoxy succinyl-L-leucylamido-(4-guanidino)butane (E-64), 0.5 mg/ml pepstatin, 0.5 mg/ml bestatin, 10 mg/ml chymostatin, and 0.1 ng/ml leupeptin. Tissue lysates (300 μg) were precleared with protein A/G-agarose and then incubated with agarose-conjugated VDR antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 4 °C overnight. The precipitates were washed with cold RIPA buffer before immunoblots using a murine monoclonal NF-κB p65 antibody (Cascade Bioscience, Winchester, MA).

Renal tissues were lysed with RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS in PBS, pH 7.4) containing 0.1 mM vanadyl sulfate and protease inhibitors (0.5 mg/ml aprotinin, 0.5 mg/ml trans-epoxy succinyl-L-leucylamido-(4-guanidino)butane (E-64), 0.5 mg/ml pepstatin, 0.5 mg/ml bestatin, 10 mg/ml chymostatin, and 0.1 ng/ml leupeptin). Tissue lysates (300 μg) were precleared with protein A/G-agarose and then incubated with either agarose-conjugated VDR antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or NF-κB p65 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 4 °C overnight. The precipitates were washed with cold RIPA buffer before immunoblots using either NF-κB p65 or VDR antibody.