Western Blot Analysis of HO-1 Expression

To analyze the expression of HO-1, BMMNCs from WT and PLC-β2-deficient mice were harvested, centrifuged, and washed twice with PBS. Protein extracts were then obtained after cell lysis using RIPA lysis buffer supplemented with protease and phosphatase inhibitors (Santa Cruz Biotechnology) and centrifugation at 15,000 rpm at −4 °C for 15 min. The protein concentrations were measured with the Pierce BCA Protein Assay Kit (Pierce, Rockford, IL) and Multimode Analysis Software (Beckman Coulter). Next, adjusted protein lysates (80 μg per sample) were separated on a 4–12 % SDS-PAGE gel, and fractionated proteins were transferred to a PVDF membrane (Bio-Rad). After blocking with 2.5 % non-fat dry milk in Tris-buffered saline containing 0.1 % Tween (TBST) for 1 h at room temperature, then washing with TBST, the membranes were incubated with a rabbit anti-HO-1 polyclonal antibody (Enzo Life Sciences, NY, USA, diluted 1:1000) overnight at 4 °C. Equal loading of proteins in all lanes was assured by reprobing with rabbit anti-β-actin monoclonal antibody (Novus Biologicals, USA, diluted 1:1000). Enhanced chemiluminescence (ECL) reagent (Amersham Life Sciences) and film (Hyperfilm, Amersham Life Sciences) were used for band visualization [19 (link), 21 (link)].