Sab4500003

Frozen kidney samples were homogenized on ice in RIPA lysis buffer containing standard protease and phosphatase inhibitors and centrifuged at 12,000 rpm and 4 °C for 5 min. The supernatant was collected, and the protein concentration was determined using a BCA assay kit. The proteins were separated by SDS-PAGE using 10% gels and then transferred to nitrocellulose membranes, which were blotted and probed overnight at 4 °C with the following antibodies: anti-JAK2 (SAB4501600, Sigma-Aldrich), anti-phospho-JAK2 (Tyr1007/1008, 07-606, Sigma-Aldrich), anti-STAT3 (SAB2104912, Sigma-Aldrich), anti-phospho-STAT3 (pTyr705, SAB4300033, Sigma-Aldrich), anti-STAT1 (AV38933, Sigma-Aldrich), anti- phospho-STAT1 (pTyr701, S2565-1VL, Sigma-Aldrich), anti- BAX (SAB3500343, Sigma-Aldrich), anti-Bcl-2 (SAB4500003, Sigma-Aldrich), anti-caspase-3 (C9598, Sigma-Aldrich) or anti-GAPDH (ab9485, Abcam). After the membranes were washed three times in TBST and incubated in HRP-conjugated secondary antibody for 1 h at room temperature, ECL luminescence solution was used to visualize the immunoreactive protein bands, and the bands were quantified using the Quantity One software package.