Lamin a c sc 376248

Protein extraction from cells was accomplished using RIPA buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS, and 10% sodium deoxycholate). Samples were separated on SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride membrane. Blotted membranes were blocked with 3% skim milk for 1 h followed by incubation with specific primary antibodies. The following antibodies were used in this study: Lamin A/C (sc-376248), GST (sc-138), green fluorescent protein (GFP) (sc-9996), p53 DO1 (sc-126), Actin (sc-47778), His (sc-8036), nuclear factor-κB (sc-372), IκBα (sc-371), and p21 (sc-397) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); α-FLAG (F3165) from Sigma (St. Louis, MO, USA); Emerin from Novocastra (New castle, UK); PUMA (4976), phospho-Erk (9101), total-Erk (9102), phospho(S15)-p53 (9286), phospho(S20)-p53 (9287), and BMI-1(5856) from Cell Signaling Technology (Danvers, MA, USA); NOXA (ALX 804-408-C100) from Enzo Life Sciences (Farmingdale, NY, USA); p16 (10883-1-AP) from Proteintech (Rosemont, IL, USA); and Histone H3k9me3 (ab8898) from Abcam (Cambridge, UK). Horseradish peroxidase-conjugated goat anti-mouse, goat anti-rabbit, and mouse anti-goat IgG antibodies (Pierce, Thermo Fisher Scientific, Inc., Rockford, IL, USA) were used as secondary antibodies.

The lysates of cell culture and mouse lungs were prepared in a lysis buffer in the presence of protease/phosphatase inhibitors, and BCA protein quantification was then performed. Denatured protein samples were loaded on 10–15% SDS-PAGE gels and transferred to PVDF membranes. After blocking the membranes with 5% skimmed milk in TBST, the membranes were incubated with the following primary antibodies: phosphorylated (p)-IκBα (cat. no. 9246), p-NF-κB p65 (3033), p-c-Jun (32740), c-Jun (9165), p-c-Fos (5348), and c-Fos (2250) (all 1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA); IκBα (cat. no. MA5-15132) and β-actin (MA5-15739) (both 1:1000; Invitrogen; Thermo Fisher Scientific, Inc., Rockford, IL, USA); NF-κB p65 (cat. no. sc-8008), iNOS (sc-651), COX-2 (sc-1747), and Lamin A/C (sc-376248) (all 1:1000; Santa Cruz Biotechnology, Inc., CA, USA). Then, the membranes were maintained with the corresponding secondary antibodies, washed with PBS, and exposed to ECL solution to visualize each band.