P erk1 2

In order to measure the levels of ERCC1 and ERK1/2, 5 × 10⁵ HROC277, HROC285, HROC370 or HROC374 cells per well were seeded in a 6-well plate, and treated by sham, 6.25 µM oxaliplatin, 5 mg/mL PRIVIGEN^® IgG, 5 mg/mL Tonglu^® IgG, 100 ng/mL IgG1 or the combination therapy indicated in the figure legends. After 36 h or 48 h, cells were harvested and western blot was performed as previously described using mouse anti-ERCC1 (Santa Cruz Biotechnology, Texas, USA, code sc-17809, dilution: 100×), mouse anti-ERK1/2 (R&D Systems, code MAB15761, dilution: 500×), rabbit anti-phospho-ERK1/2 (p-ERK1/2, R&D Systems, code MAB1018, dilution: 1000×) and mouse anti-β-actin antibody (Sigma-Aldrich, code A5441, dilution: 20,000×), followed by the secondary antibodies: peroxidase-linked anti-rabbit (Cell Signaling Technology, code 7074, dilution: 5000–10,000×) or anti-mouse antibodies (Sigma-Aldrich, code A9044, dilution: 20,000–60,000×). Proteins were visualised as previously described.^{29 (link)}

RAW264.7 cells were cultured in different concentrations of CFP (1.875, 3.75, 7.5, or 15 μg/mL) or 1 μg/mL of LPS. Using RIPA buffer (Tech & Innovation, China), the cells were lysed and protein was extracted. Protein concentration was measured using the Pierce™ BCA Protein Assay kit (Thermo Scientific, USA). After resolving the proteins using SDS-polyacrylamide gel electrophoresis (SDS-PAGE), they were transferred on to a polyvinylidene fluoride (PVDF) membrane. Western blotting was performed as described previously [28 (link)]. using antibodies specific for p-NF-κB p65 (R&D systems), p-IκBα (Cell Signaling Technology), p-p38 (R&D systems), p-ERK1/2 (R&D systems), p-JNK (R&D systems), and α-tubulin (Abcam). Target proteins were detected using the Pierce® ECL Plus Western Blotting Substrate (Thermo Scientific, USA). Imaging was performed using the ChemiDoc XRS+ imaging system (Bio-Rad) and protein band intensities were analyzed using the ImageLab software (version 4.1, Bio-Rad).

Colon-specific (L₆-S₂) spinal cord samples were homogenized in lysis buffer (Invitrogen). Protein concentration was determined by BCA Protein Assay Kit (Invitrogen). Protein samples were separated on instant SDS–PAGE gel and transferred to PVDF membranes. Blots were blocked with 5% milk and incubated overnight at 4°C with an antibody against GFAP (1 : 500, abcam), P2Y₁ (1 : 500, abcam), pERK1/2 (1 : 200, R&D Systems), and PKC (1 : 300, R&D Systems). The anti-rabbit IgG concentration of goat was 1 : 1000. The blots were further incubated with anti-rabbit IgG secondary antibodies for 2 h at room temperature and captured by Imaging Lab. The intensity of the bands was quantified using ImageJ (NIH, Bethesda, MD).