Ab45420

The protein expression levels were measured by western blot. In brief, total protein was obtained using the specific protein extraction kit (BestBio Institute of Biotechnology, Wuhan, China). The amounts of total protein were quantified by the BCA assay (Keygen Institute of Biotechnology, Nanjing, China). Protein was resolved by 6–15% SDS/PAGE and transferred onto poly(vinylidene difluoride) membranes (EMD Millipore, Billerica, MA, USA), then blocked with 5% nonfat milk. Primary antibodies, including P‐gp (1 : 1000; ab170904; Abcam, Cambridge, UK), STAT3 (1 : 1000; ab76315; Abcam), IL‐8 (1 : 1000; ab18672; Abcam), IL‐23 (1 : 1000; ab45420; Abcam), VEGF (1 : 1000; ab46154; Abcam), p‐STAT3 (1 : 1500; 9145; Cell Signaling Technologies, Danvers, MA, USA), MRP1 (1 : 1000; 72202; Cell Signaling Technologies), IL‐1β (1 : 1000; 12703; Cell Signaling Technologies) and p‐NF‐κB (1 : 800; sc‐136548; Santa Cruz Technology, Santa Cruz, CA, USA), were added and incubated overnight at 4 °C. The anti‐IgG secondary antibodies (ab205718, ab190475; Abcam) were subsequently applied to the membranes and incubated for 2 h. Immunoreactive signals were revealed by the enhanced chemiluminescence detection system (GE Healthcare, Muenchen, Germany). imagej software (National Institute of Health, Bethesda, MD, USA) was applied to analyze protein expressions.

Total protein was extracted using RIPA lysis buffer (cat. no. R0020; Beijing Solarbio Science & Technology Co., Ltd.). Equal quantities of denatured protein (30 μg) were separated by 10% or 12.5% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (cat. no. ipvh00010; Sigma‐Aldrich, Merck KGaA). After blocking in 5% skim milk for 60 min, the membranes were incubated with primary antibodies against RORγt, IL‐23, and IL‐17A (all 1:1000 dilution, cat. no. ab113434, ab45420, and ab13556, Abcam) and TLR4 antibody (1:500 dilution, cat. no. sc‐293072; Santa Cruz) at 4°C overnight and then incubated with the corresponding peroxidase‐conjugated goat anti‐rabbit IgG antibodies (1:10,000 dilution, cat. no. ZB‐2306, ZSGB‐BIO) for 1 h at room temperature. β‐actin antibody (1:10,000 dilution, cat. no. ab179467, Abcam) was used to confirm equal protein loading in each lane. The protein bands were detected by an ECL kit (cat. no. MA0186, Dalian Meilun Biology Technology Co., Ltd.) and analyzed with ImageJ software v 4.1 (National Institutes of Health).