Ab133369

Cells lysates were prepared using lysis buffer (150 mM NaCl, 1% tritonX-100, 0.5 % sodium deoxycholate, 0.1% SDS, 50 mM tris pH 8) containing protease inhibitor and phosSTOP phosphatase inhibitor cocktail (Roche). Protein concentration was measured using BCA protein assay (Pierce) for western blot analysis. Primary antibodies were purchased from Santa Cruz for transferrin (sc-374441), TfR1 (sc-32272), DRD2 (sc-5303), DRD3 (sc-136170), DRD4 (sc-136169), DRD5 (sc-376088), TH (sc-25269), PAH (sc-271258), and DDC (sc-293287); from Cell Signaling Technology for SOX2 (#3728), CD44 (#3570), pSrc (#6943), Src (#2108), pSTAT3 (#9131), STAT3 (#9139), pERK (#9101 S), ERK (#9102 S), and PARP (#9542 S); from Novus Biologicals for HIF1α (NB100-449) and DRD1 (NBP2-66807); from Abcam for 4-HNE (ab46545), β-actin (ab6276), and HFE (ab133369). Secondary antibodies include HRP conjugated anti-mouse IgG (ab6728) from Abcam and anti-rabbit IgG (G-21234) from Innovative Research. Antibody concentration was 1:1000 for DRD1, DRD2, DRD3, DRD4, DRD5, TH, PAH, and DDC; 1:2000 for transferrin, TfR1, SOX2, CD44, pSrc, Src, pSTAT3, STAT3, pERK, ERK, PARP, HIF1α, and HFE; 1:3000 for 4-HNE; 1:5000 for β-actin and secondary antibodies.

The tissues were from single patient. The immunohistochemistry (IHC) staining was performed according to the manual instructions (#13079, Cell Signaling Technology, MA, USA). Anti-SURF4 primary antibody (ab133369, Abcam company, Shanghai, China) and corresponding secondary antibody (rabbit) were used. The fluorescence microscope was used for imaging.