Rabbit anti gapdh fl 335

Whole cell extracts were prepared by solubilizing cells in RIPA buffer supplemented with Complete Mini protease inhibitor cocktail (Roche Diagnostics; Indianapolis, IN, USA), followed by a freeze-and-thaw cycle, and clarification with centrifugation. Protein concentrations were quantified using the Micro BCA assay (ThermoFisher). Equal amounts of protein (25 μg) were resolved using 10% SDS-PAGE and immunoblotted according to standard protocols. The membranes were blocked with 5% non-fat milk prepared in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 hr, followed by incubation with primary antibodies prepared in TBST at 4 °C overnight. Primary antibodies used in this study include: CD36 (SMφ) (Santa Cruz Biotechnology), ATGL (30A4) (Cell Signaling; Danvers, MA, USA), PPARγ (C26H12) (Cell Signaling), and phospho-β-catenin (D2F1) (Cell Signaling). Secondary antibody was peroxidase-conjugated goat anti-mouse or rabbit IgG (1:10000, Jackson ImmunoResearch; West Grove, PA, USA) prepared in TBST. The membranes were incubated at room temperature for 1 hr. Protein bands were visualized using Immobilon Western chemiluminescent HRP substrate (EMD Millipore; Billerica, MA, USA). The blots were stripped and reprobed with rabbit anti-GAPDH (FL-335) (Santa Cruz Biotechnology) to ensure equal loading.

Protein extracts (20 µg) were analyzed by immunoblotting. The primary antibodies used to probe blots were goat anti-WNV NS3 (R&D Systems), rabbit anti-ISG49 (IFIT3; kindly provided by G. Sen), rabbit anti-GAPDH (FL-335; Santa Cruz), and rabbit anti-STAT1 (Cell Signaling) antibodies. The secondary antibodies used included peroxidase-conjugated goat anti-rabbit and donkey anti-goat antibodies (Jackson ImmunoResearch, Inc.). Densitometry analysis was performed with Image Studio Lite software (LI-COR).