Anti p erk1 2

Western blotting analysis was carried out as previously described [25 (link)]. The following antibodies were used: anti-Bcl-2 (#ab182858), anti-Bax (#ab182733), anti-Bcl-xl (#ab32370), anti-p-P65 (#ab76302), anti-P65 (#ab32536), anti-p-IκBα (#ab133462), anti-IκBα (#ab32518) (Abcam Cambridge, MA, USA), anti-FXR (#sc-25309), anti-TLR4 (#sc-293072) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p-ERK1/2 (#4370), anti-ERK1/2 (#4695), anti-p-P38 (#4511), anti-P38 (#8690), anti-p-JNK1/2 (#4668), anti-JNK1/2 (#9252), anti-CCL2 (#66272-1-Ig), and anti-β-actin (#66009-1-Ig) (Proteintech, Wuhan, China).

Total proteins were extracted from cells and tissues. The cells and tissues were lysed with 20 μL of RIPA Buffer (Roche Molecular Biochemicals, Switzerland), and protein concentrations were detected using the BCA kit (Beyotime, China), according to the manufacturer’s guidelines. Protein samples (20~40 μg) were separated on an SDS-PAGE gel and transferred to nitrocellulose membranes. After blocking the membranes with 5% skim milk in phosphate-buffered saline (PBS) for 1 h at room temperature, the membranes were probed with anti-IL-21 (Abcam, UK), anti-claudin-5 (GeneTex, USA), anti-p-NF-κB (Santa Cruz Biotechnology, USA), anti-NF-κB (Santa Cruz Biotechnology, USA), anti-p-ERK1/2 (Proteintech, USA), anti-ERK1/2 (Proteintech, USA), anti-p-JNK (Santa Cruz Biotechnology, USA), anti-JNK (Proteintech, USA), anti-p-P38 (Santa Cruz Biotechnology, USA), anti-P38 (GeneTex, USA) and GAPDH (Zhongshanjinqiao, China) antibodies at 4°C overnight. The nitrocellulose membranes were washed with PBST (PBS containing 0.5% Tween 20) three times for 7 min each, which was followed by the incubation with a fluorescence-labeled secondary antibody (IRDye700/800 mouse and rabbit antibodies) (Santa Cruz Biotechnology, USA). Protein levels were determined using an Odyssey infrared scanning system (LI-COR Biosciences, USA), and the band intensities were quantified using ImageJ software.

Cells were lysed with RIPA buffer (Beyotime, China) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) for 10 min on ice and centrifuged at 14,000 × g. The supernatants were collected, and whole cell extracts were separated by 10% SDS-PAGE gel and transferred onto a PVDF membrane (GE, USA). The membranes were blocked with 5% skimmed milk (BD, USA) in TBST buffer and incubated with primary antibodies at 4 °C overnight. Primary antibodies used were anti-IRS2 (1:1,000, Proteintech), anti-CCND1 (1:1,000, Servicebio, China), anti-ERK1/2 (1:1,000, CST, USA), anti-p-ERK1/2 (1:1,000, Proteintech, China), and anti-β-actin (1:1,000, Sigma, USA). After washing with PBS five times, the membranes were incubated with near-infrared fluorescent anti-mouse (1:50,000, LI-COR, USA) or anti-rabbit (1:25,000, Jackson, USA) secondary antibodies for 1 h at room temperature. Fluorescent signal was detected by Odyssey Near-infrared fluorescence imaging system (LI-COR, USA).