Hrp conjugated goat anti rabbit igg sc 2004

Epididymal and inguinal adipose tissues were lysed in ice-cold lysis buffer (50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 0.02% Sodium azide and 1% Triton X-100) containing protease inhibitors (phenylmethylsulfonyl fluoride and aprotinin). Lysates were centrifuged at 12,000 rpm for 20 min at 4°C and the resulting supernatants (10 μg) were subjected to electrophoresis on 10% polyacrylamide gels. The separated proteins were transferred to PVDF membrane (Millipore, Billerica, MA, USA). Membranes were incubated with primary antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The primary antibodies were anti-VEGF antibody (sc-507), anti-MMP-2 antibody (sc-10736) and anti-MMP-9 antibody (sc-10737) (1:200 dilution). After incubating with HRP-conjugated goat anti-rabbit IgG (sc-2004, Santa Cruz) (1:2500 dilution) as a secondary antibody, blots were visualized using an ECL Western blot detection system (Intron, Daejeon, Korea).

Whole-cell extracts (10⁶ cells per experimental point) were prepared using RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and 1 mM EDTA) supplemented with protease inhibitors (Sigma-Aldrich). Proteins were separated using SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. Membranes were incubated with a rabbit polyclonal antibody to HIV-1 Tat (ab43014, Abcam, Cambridge, UK) and a rabbit monoclonal antibody to β-actin (Cell Signaling Technology #4970, Danvers, MA, USA), which served as a loading control. HRP-conjugated goat anti-rabbit IgG (sc-2004, Santa Cruz, CA, USA) was used as a secondary antibody. Protein levels were visualized using the ECL LumiGLO detection kit (Upstate, Biotechnology UBI).