Protein Extraction and Detection Protocol

Samples were lysed using radioimmunoprecipitation assay buffer (RIPA) lysis buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris pH 8.0, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS) supplemented with 1 mM dithiothreitol and protease inhibitors. The lysates were separated by SDS-PAGE and analyzed using standard western blotting procedures (Khan et al., 2019 (link)). Antibodies: human NR4A1 (ab153914, 1:1,000; Abcam), mouse NR4A1 (14-5965-82, 1:1,000; Invitrogen), NR4A2 (AV38753, 1:1,000; Sigma-Aldrich), NR4A3 (TA804893, 1: 1,000; Thermo Fisher Scientific), VHL (68547, 1:1,000; Cell Signaling), β-actin (13E5, 1:5,000; Cell Signaling), β-tubulin (9F3, 1: 5,000; Cell Signaling), and GAPDH (D16H11, 1: 5,000; Cell Signaling) were employed for protein detection.