T5030

The protein samples were separated by SDS-PAGE and transferred to polyvinylidene fluoride membrane, followed by blockage with 5% bovine serum albumin (Sigma, B2064) in Tris-buffered saline (Sigma, T5030) containing 0.1% Tween 20 (Sigma, 93773) (TBST). The membranes were then incubated with primary antibodies at 4°C overnight. The primary antibodies included anti-NF-κB p65 (Abcam, ab16502), anti-Nrf2 (Abcam, ab31163), anti-GAPDH (glycer-aldehyde-3-phosphate dehydrogenase) (Abcam, ab9485), and anti-Lamin B1 (Proteintech, 66095-1-Ig) antibodies. After three washes with TBST, the membranes were incubated with HRP-goat-anti-rabbit (Abcam, ab6721) or HRP-goat-anti-mouse (Abcam, ab6789) secondary antibodies at room temperature for 1 h, followed by visualization using an enhanced chemiluminescence (ECL) system (Thermo Scientific, 32132). The images of protein bands were photographed using a ChemiDoc MP device (Bio-Rad, Hercules, CA, USA) and analyzed using ImageJ software.

Briefly, protein concentration was determined using the Pierce BCA Protein Assay Reagent (Thermo Fisher Scientific, 23228, Waltham, MA, USA). From each sample, 30 μg protein was resolved by SDS-PAGE on Tris-glycine gels and transferred to polyvinylidene fluoride membranes. Membranes were blocked with 5% bovine serum albumin (B2064, Sigma-Aldrich, St. Louis, MO, USA) in Tris-buffered saline (T5030, Sigma-Aldrich, St. Louis, MO, USA) containing 0.1% Tween 20 (93773, Sigma-Aldrich, St. Louis, MO, USA) (TBST) and incubated with primary antibodies overnight at 4 °C. Membranes were washed three times for 5 min with TBST, incubated with either HRP-goat-anti-mouse (Abcam, ab6789, Cambridge, UK) or HRP-goat-anti-rabbit (Abcam, ab6721, Cambridge, UK) secondary antibodies for 1 h at room temperature. Blots were visualized using enhanced chemiluminescence (ECL from GE Healthcare, Braunschweig, Germany). Bands were quantified using Quantity One Software (Biorad, Hercules, CA, USA).

Cells were lysed with IGEPAL CA-630 buffer (50 mM Tris-HCl, pH 7.4, [T5030, Sigma-Aldrich, St. Louis, MO, USA], 1% IGEPAL CA-630 [I8896, Sigma-Aldrich, St. Louis, MO, USA], 10 mM EDTA, 150 mM NaCl, 50 mM NaF, 1 μM leupeptin [L5793, Sigma-Aldrich, St. Louis, MO, USA], and 0.1 μM aprotinin [SRE0050, Sigma-Aldrich, St. Louis, MO, USA]). Lysates were incubated with primary antibodies at 4 °C for 16 h and then incubated with protein G sepharose (GE healthcare, Uppsala, Sweden) at 4 °C for 3 h. Immunoprecipitated proteins were eluted at 95 °C for 2 min with 30 μL of 2× sample buffer (0.1 M Tris-HCl, pH6.8, 0.2 M DTT, 4% SDS, 20% glycerol, 0.2% bromophenol blue, and 1.43 M β-mercaptoethanol) and then used for immunoblotting.