Photo image system

Cells were washed once with PBS and lysed in lysis buffer (Cell Signaling Technology, Danvers, MA, USA) containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM Na₂-EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 mg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride. The protein concentrations were determined using the Bradford Protein Assay Kit (BioRad, Hercules, CA, USA). Proteins were separated in a 12% sodium dodecyl sulfate-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Millipore, USA). After blocking the membrane with Tris-buffered saline-Tween 20 (TBS-T, 0.1% Tween 20) and 5% nonfat dried milk for 1 h at room temperature, the membrane was washed twice with TBS-T and incubated with primary antibody (ICAM1, VCAM1, MMP-2, MMP-9, p-ERK, ERK, p-NF-κB, NF-κB, and β-actin (Santa Cruz Biotechnology: Dallas, TX, USA; Abcam: Cambridge, UK; Cell Signaling: Danvers, MA, USA)) overnight at 4 °C. Next, the membrane was washed three times with TBS-T for 10 min and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies. After washing, the bands were detected with an enhanced chemiluminescence reagent (Santa Cruz Biotechnology). The band intensities were quantified using a Photo-Image System (Molecular Dynamics, Caesarea, Israel).

Cells were washed once in PBS and lysed in RIPA lysis buffer (P0013B; Beyotime, Shanghai, China) at 4°C. Proteins were denatured by boiling. Protein concentrations were determined using the Enhanced BCA Protein Assay kit (P0010S; Beyotime). Protein samples were separated in a 12% SDS-polyacrylamide gel and transferred to PVDF membranes (Immobilon-P membranes, Millipore, USA). Membranes were blocked with Tris-buffered saline/Tween 20 (TBST, 0.1% Tween 20) containing 5% bovine serum albumin (BSA) for 1 h and then incubated with the appropriate primary antibody overnight at 4°C. After three 10-min washes with TBST, membranes were incubated for 1 h at 24°C with the appropriate horseradish peroxidase-conjugated secondary antibody. After extensive washing, immunoreactive bands were detected by the BeyoECL Plus reagent (P0018; Beyotime) using a Photo-Image System (Molecular Dynamics, Sunnyvale, CA, USA). Immunoblotting was performed with the following primary antibodies: phosphorylated NRF2 Ser40 (pNRF2 Ser40) (ab76026; Abcam), NRF2 (ab62352; Abcam), GCLC (ab53179; Abcam), GGT1 (ab175384; Abcam), NQO1 (ab34173; Abcam), HO1 (ab13243; Abcam), Retinoblastoma (RB) (ab24; Abcam), p53 (ab1101; Abcam), p21 (ab109520; Abcam), p16 (ab51243; Abcam), CRIF1 (sc-134882; Santa Cruz), PKC-δ (sc-213-G; Santa Cruz), and β-actin (sc-8432; Santa Cruz).