Lamin a c and gapdh

Cells or tissues were lysed by a freeze-thaw cycle in ice-cold lysis buffer [1% Nonidet P-40, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA (pH 8.0), 1 mM NaF] followed by sonication in the presence of a protease inhibitor cocktail (Roche). Samples were centrifuged for 20 min at 14,000 × g at 4 °C, and the supernatants were collected. Protein concentration was measured using a BCA Protein Assay Kit (Pierce). For each sample, 20–50 μg of proteins were prepared in 4X NuPAGE buffer (Invitrogen) and heated to 95 °C for 5 min prior to loading on a 4–12% SDS-PAGE gel (Invitrogen). After separation and transfer of the proteins to PVDF membranes (Amersham), the membranes were blocked with 5% nonfat dry milk and probed with appropriate primary antibodies followed by HRP-conjugated secondary antibodies (Cell Signaling) at a dilution of 1:5000. Bands were visualized using SuperSignal Chemiluminescent Substrate (Pierce) exposed on ECL Film (ISC BioExpress), or CCD camera. The following antibodies were used: p16^INK4a, P-S6 (Cell Signaling), P-(Ser139)-H2AX (H2AXγ) (Cell Signaling), HO-1 (Enzo Laboratories), β-Actin (Sigma Aldrich), lamin A/C and GAPDH (Cell Signaling Technologies).

RAW 264.7 murine macrophage cells were plated in 6-well plates at the density of 1 × 10⁶ cells/well and grown for 12 h for adherence. The cells were pretreated with Cath-MH (0, 1, 2, 4, and 8 μM) for 1 h and then stimulated with LTA (10 μg/ml) or LPS (100 ng/ml) at 37°C for 30 min in 5% CO₂. After that, the cells were lysed with RIPA lysis buffer (Beyotime Biotechnology, China) and proteins were extracted using commercial kit (Cayman Chemical, United States) in light of the manufacturer’s recommendations. Primary antibodies of phospho-ERK/ERK, phospho-JNK/JNK, phosphor-p38/p38, NF-κB p65, Lamin A/C and GAPDH (1: 1,500, Cell Signaling Technology, United States) and horseradish peroxidase conjugated secondary antibodies (1: 2,000, Cell Signaling Technology, United States) were applied in western blot analysis. All experiments were repeated three times.