Nf kb

Protein extraction was performed using the Nuclear and Cytoplasmic Protein Extraction kit (Thermo Scientific, Pierce Biotechnology, IL, USA) according to the manufacturer’s instructions. The samples were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto PVDF membranes (Bio-Rad, Hercules, CA, USA). Subsequently, the membrane was blocked in TBST (20 mM Tris base pH 7.6, 150 mM NaCl, 0.1% Tween-20) containing 5% non-fat milk for 4 h at room temperature. Then, the membrane was incubated with NF-kB (Invitrogen, Cat number MA5-15160) and Lamin B1 (Invitrogen, Cat number 33-2000) primary antibodies that were added to the blocking solution at 3 µg/mL, and the membrane was incubated at 4 °C overnight. After overnight incubation, the membrane was washed and then incubated with goat anti-rabbit immunoglobulin G HRP conjugated secondary antibodies (Invitrogen, Cat number G-21234) and goat anti-mouse immunoglobulin G HRP conjugated secondary antibodies (Invitrogen, Cat number PA1-86015) for an additional hour at room temperature. After washing three times with TBST (30 min each wash), proteins were detected by chemiluminescence and protein bands were analyzed by densitometry using ImageLab software (Bio-Rad 6.1, Hercules, CA, USA).

EpASCs proteins were extracted by sonication using the Lysis buffer (Sigma-Aldrich) containing a cocktail of protease and phosphatase inhibitors. Cell-lysates were centrifuged (13,000g, 4°C, 15 min) and the supernatant was saved. The protein concentration was determined by the Bradford method. Aliquots (40 μg protein) from each sample were first heat-inactivated (95°C, 10 min) in denaturing buffer [10% glycerol, 1% SDS, 1% β-mercapto-ethanol, 0.01% bromophenol blue, 10 mMTris-HCl (pH 6.8)], and then electrophoresed on 12% polyacrylamide gel. Protein bands were blotted onto Immobilon-P (Millipore) already pre-blocked for unspecific protein binding (1 hour with 5% skimmed milk in TBS-T buffer, pH 7.6). Blots were washed extensively three times in 0.1% Tween-20 in TBS for subsequent incubation with primary antibodies (overnight, 4°C). The primary antibody for Nrf2 (Sigma), NFkB (Invitrogen), CK10, CK14, (Santa Cruz Biotechnology), PCNA, P38, p63, TNF-α (Cell Signaling)was used in 1:5,000 dilution and the HRP conjugated secondary antibody (Cell Signaling) was used in 1:10,000 dilution. The probed membranes were incubated with substrate (5 min, RT) and developed with Enhanced Chemiluminescence (ECL) kit (Thermo, USA). Immunoblots were stripped and re-probed with β-actin antibody (Sigma-Aldrich) for loading correction.

Cells were harvested and lysed in radioimmune precipitation assay (RIPA) buffer, 0.5M EDTA and proteases and phosphatase inhibitors cocktail total protein quantified by BCA protein assay kit (Pierce). For Western blots, 30 mg of protein extract/lane were electrophoresed, transferred to nitrocellulose membrane (Invitrogen) and incubated overnight with each of the following primary mouse monoclonal antibody: RASAL2 (1:2000; sc-390605; Lot# 2516); NF-kB (1:500; sc-8008; Lot# H1220); N-RAS (1:1000; sc-31; Lot# JQ520); TNFα (1:500; sc-515766 Lot# 17020); C-myc (1:500; sc-40; Lot# J0220); AR (1:1000; sc-7305; Lot# J2920); PTEN (1:1000; sc-7974; Lot# 10420). The GAPDH antibody (1:5000; sc-32233; Lot# J2020) was used as an internal loading control. Membranes were washed and incubated with anti-mouse secondary antibody (1:2500; sc-2005). All the antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). The antigen-antibody reaction was visualized using an enhanced Chemiluminescence (ECL) assay (Bio-Rad; Hercules, CA, USA) and image the membrane using digital imager/chemidoc MP imaging system (Bio-Rad). A densitometry program using ImageJ (https://imagej.nih.gov/ij/), was used to quantify bands in the western blot and protein expression level displayed as ratio of each protein to the GAPDH protein level. The data are a representative of triplicate experiments.