Lox 1

A standard Western blotting protocol was used as described previously [5 (link),25 (link)] with antibodies against TGFβ (1:600), matrix metalloproteinase 2 (MMP2, 1:200) (Santa Cruz, CA, USA); connective tissue growth factor (CTGF, 1:500) (Biovision, Mountain View, CA, USA), LOX-1 (1:1000) (R&D System), bone morphogenetic protein-7 (BMP-7, 1:5000) (AbDSerotec, Minneapolis, MN, USA), nuclear factor-kappa B (NFκB, 1:1000), its inhibitor kappa B alpha (IκBα, 1:200), Phospho-P38 (1:1000) (Millipore, Billerica, MA, USA), NADP(H) oxidase subunit Gp91phox (1:500, BD Transduction Laboratories, France). Loading controls were β actin (1:3000, Sigma Aldrich, France) or P38 (1:1000, Millipore). Appropriate HRP-coupled secondary antibodies (1:5000 to 1:10 000, GE Healthcare, France) were used to detect the band by chemiluminescence with ECL plus (GE Healthcare, France). Intensities of the protein bands were determined and quantified using AlphaEase FC software (Alpha Innotech Corporation, San Leandro, CA).

Spautin-1 and MG132 were purchased from Selleckchem (Houston, TX, USA). Control (SC-37007) and USP10 (SC-76811) siRNAs were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Human dil-labeled oxLDL (YB-0010) oxidized low density lipoprotein (oxLDL) (YB-002) were from Yiyuan Biotechnologies (Guangzhou, China). Antibodies are as follows: anti-GAPDH (#5174), anti-USP10 (#8501), anti-ubiquitin (#3936), anti-K48-linkage Specific Polyubiquitin (#12805), anti-CD36 (ab13365), ABCA1 (#96292), ABCG1 (ab52617), SR-B1 (ab217318), SR-A (ab123946). Lox-1 was from R&D system. Oil Red O was from Sigma-Aldrich. CO-IP were obtained from Novus biologicals (USA).

Using Western blot analysis, we examined the protein expression of LOX-1 (antibody from R&D Systems, Minneapolis, MN) and TNF-α (antibody from GeneTex Inc., Irvine, CA) in BAECs treated with 30 μg/mL LDL from male db/db mice or PBS in the presence or absence of pretreatment with 17β-estradiol (10 nM) or genistein (100 nM), as described previously [19 (link)].