FIG. 2.

Anti-PLA2L Western blot of lysates of various cell lines and transfectants. The Western blot was blocked overnight at 4°C in 2% BLOTTO (5% skim milk powder in phosphate-buffered saline), washed three times for 1 h each time in TBST (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.05% Tween 20 [Sigma]), and hybridized to rabbit antiserum in 2% BLOTTO for 1 h. All washing and hybridization steps, unless noted otherwise in the text, were carried out at room temperature with constant agitation. The Western blot was then washed four times for 30 min each time in TBST. The secondary antibody consisted of horseradish peroxidase-conjugated AffiniPure goat anti-rabbit immunoglobulin G (Jackson Immunoresearch Laboratories), which was used at an approximately 1:8,000 dilution in TBST for a 50-min incubation. The Western blot was then washed four times for 30 min each time in TBST and visualized with a Renaissance enhanced-chemiluminescence kit (Dupont, NEN). Molecular weights (MW) are noted in thousands.