Pan actin antibody

Primary peritoneal macrophages were harvested from HO-1^+/+apoE^−/− and HO-1^−/−apoE^−/− mice according to the method described previously [41 ]. Briefly, 4% Brewer thioglycollate (BD Bioscience) medium was injected into the peritoneal cavity of 6-8 weeks old mice. Four days after injection, mice were euthanized by CO₂ inhalation. Primary peritoneal macrophages were harvested and plated on culture dishes with RPMI 1640 medium containing 10% FBS. After 2 h incubation, nonadherent cells were removed by washing with RPMI medium. Adherent cells were then used for experiments. To determine the effect of different mediators on MMP9 activity, macrophages were treated with 10 μmol/L angiotensin II or 100 ng/mL eotaxin (ProSpec). At different time points following stimulation, conditioned medium was collected for measuring MMP9 activity and cytokine productions. Total proteins were prepared from macrophages for Western blot analysis to detect MMP9 (Abcam, 1:1,000) and HO-1 (Enzo, 1:500). Equivalent loading was verified by incubating blots with a pan-actin antibody (Millipore, 1:80,000).

Approximately 8 weeks old HO-1^+/+apoE^−/− and HO-1^−/−apoE^−/− mice were sacrificed by an overdose of tribromoethanol solution (500-750 mg/kg) by IP injection. Primary VSMCs were then isolated from aortas and cultured in DMEM as described [40 (link)]. Cells of passages 3-6 were used for experiments. To determine the effect of oxidant on cell viability, HO-1^+/+apoE^−/− and HO-1^−/−apoE^−/− VSMCs were plated in 24-well plates (4×10⁴ cells/well), serum starved in quiescent medium (0.2% FBS) for 36 h, then stimulated with increasing concentrations of H₂O₂ for 24 h. MTT assays were then performed and cell viability presented as percentage of control without H₂O₂. To evaluate the effect of angiotensin II on MMP2 activity, HO-1^+/+apoE^−/− and HO-1^−/−apoE^−/− VSMCs were serum starved, stimulated without or with different concentrations of angiotensin II for 24 h, and the conditioned medium collected. Conditioned medium was then concentrated and subjected to zymography to determine MMP2 activity. Total proteins were isolated from VSMCs for Western blotting. To determine MMP2 and HO-1 protein expressions, the blots were incubated with MMP2 (Abcam, 1:500) and HO-1 (Enzo, 1:500) antibodies, respectively. The blots were subsequently probed with a pan-actin antibody (Millipore, 1:80,000) to verify equivalent loading.

G-actin and F-actin fractions in cultured cells were separated using the G-Actin/F-Actin In Vivo Assay Kit according to the manufacturer's instructions (BK037, Cytoskeleton, Denver, CO). Briefly, cells were lysed using lysis and actin stabilization buffer supplemented with protease inhibitor cocktail and 1 mM ATP, scraped into Eppendorf tubes, and passed through a 25G syringe. Cell lysates were incubated at 37°C for 10 min and centrifuged at 350 g for 5 min to remove the debris. Supernatants were then transferred into ultracentrifuge tubes (349622, Beckman Coulter, Brea, CA) and centrifuged at 100,000 g at 37°C for 1 h. The resultant supernatants were designated as G-actin fractions. Then an equal volume of F-actin depolymerization buffer was added to the pellet and incubated on ice for 1 h with pipetting every 15 min. Resulting samples were designated as F-actin fractions. Each sample was then added 5× SDS sample buffer and boiled for 5 min to denature. An equal volume of samples from each fraction was loaded onto SDS-PAGE gels and subjected to western blotting. The amount of actin in G-actin or F-actin fraction was determined using a pan-actin antibody (A2103, Sigma-Aldrich) to detect all actin isoforms. Band intensity was quantified by densitometry and the G-actin:F-actin ratio was plotted.