Immun star hrp luminol enhancer and peroxidase buffer

Macrophages infected or not with L. amazonensis axenic amastigotes were washed twice with cold PBS, scraped from the dish, collected by centrifugation (335 g) for 10 min at 4°C, lysed in 30 μl RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.50% sodium deoxycholate, 0.10% SDS) with protease inhibitors (Roche), and centrifuged at 14000 g for 15 min at 4°C to remove insoluble material. Lysates were assayed for protein content (Pierce BSA Protein Assay kit), 60 μg per sample were mixed with SDS sample buffer at room temperature for 30 min, separated by SDS-PAGE (without sample boiling) and transferred to a PVDF membrane (Millipore). Membranes were incubated overnight with 1:10000 mouse anti-paxillin antibody (BD Transduction Laboratories), 1:1000 rabbit anti-paxillin (pY¹¹⁸) (Invitrogen), 1:1000 rabbit anti-FAK (pY³⁹⁷) (Abcam) or 1:5000 anti-actin (Sigma-Aldrich) in blocking buffer (PBS 3% milk, 0.1% Tween), followed by HRP-coupled anti-rabbit IgG or anti mouse IgG secondary antibodies (Amersham Biosciences). Blots were developed using Immun-Star HRP Luminol Enhancer and peroxidase buffer (Bio-Rad) and detected with a Fuji LAS-3000 Imaging System and Image Reader LAS-3000 software. Digital quantifications of chemiluminescence were performed using Image J software.