Af5195

Total protein was extracted from 80 mg samples of colon tissue with 400 μL of radioimmunoprecipitation assay buffer (Solarbio, Beijing, China). Proteins (10 μg) were separated using SDS-polyacrylamide gel electrophoresis (10% gels) and then were transferred to polyvinylidene fluoride membranes. The membranes were blocked in 5% skim milk for 2 hours and incubated with one of the following primary antibodies overnight at 4°C : TLR4 (1 : 1000, 19811-1-AP, Proteintech, Wuhan, China), MyD88 (1 : 2000, AF5195, Affinity Bioscience, Beijing, China), NF-κB (1 : 2000, 10745-1-AP, Proteintech, Wuhan, China), p–NF–κB (1 : 2000, AF3387, Affinity Bioscience, Beijing, China), cleaved caspase-3 (19677-1-1AP, 1 : 2000, Proteintech, Wuhan, China), Bcl2 (1 : 3000, 26593-1-AP, Proteintech, Wuhan, China), Bax (1 : 12,000, 50599-2-Ig, Proteintech, Wuhan, China), or β-actin (1 : 50,000, 6609-1-Ig, Proteintech, Wuhan, China). On the next day, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (H + L) (1 : 10000, Affinity Bioscience, Beijing, China) or goat anti-mouse IgG (H + L) (1 : 10000, SA00001-1, Affinity Bioscience, Beijing, China) for 2 hours at room temperature. An ECL Chemiluminescent Kit (Beyotime, Shanghai, China) was utilized to detect the protein bands. Gel-Pro Analyzer software (Version: 4.0.0.4) was used to quantify the band intensities.

Samples were lysed in a RIPA buffer in the presence of protease and phosphatase inhibitors. Proteins were resolved on 5–15% SDS‐polyacrylamide gels and transferred to PVDF membranes. After blocking with 5% BSA, membranes were incubated overnight at 4 °C with the appropriate primary antibodies. After washing with TBS‐0.1% tween‐20 for 5 min, the membranes were soaked in horseradish peroxidase‐conjugated secondary antibodies (1:5000 dilution) for 2 h. Membranes were washed again before visualization. The Fdbio‐Dura ECL Kit (Fudebio‐tech, China) was used for protein band visualization in photographic films (Biostep, Germany). The following antibodies were used for western blot analysis: p‐Nrf2 (Ab76026, Abcam), NQO1(Ab80588, Abcam), HO‐1(Ab68477, Abcam), BAX (Ab32503, Abcam), Bcl‐2 (Ab182858, Abcam), TLR4 (AF7017, Affinity), MyD88 (AF5195, Affinity), IL‐1β (AF5103, Affinity), IL‐6 (DF6087, Affinity), β‐actin (AF7018, Affinity), goat anti‐rabbit IgG (H+L) HRP (S0001, Affinity) were used as secondary antibodies.

The levels of NF-κB, TLR2, and MYD88 in lung tissues were tested using immunohistochemistry. Paraffin-embedded rat lung tissue sections were deparaffinized to water, antigen retrieval was performed in citrate buffer of pH 6.0, and microwave treatment was performed for 20 min. The sections were placed in a 3% H₂O₂ solution, incubated at room temperature in the dark for 25 min, and serum-blocked for 30 min. The blocking solution was gently shaken off, and the primary antibodies MyD88 antibody (AF5195, 1 : 125, Affinity), NF-kBp65 antibody (AF5006, 1 : 125, Affinity), and TLR2 antibody (DF7002, 1 : 125, Affinity) were added dropwise to the section and incubated overnight at 4°C. The sections were placed in PBS and shaken 3 times with bleach. After the sections were dried, the secondary antibody (HRP) (S0010, 1 : 200, Affinity) was added dropwise and incubated for 50 min. Then, the slides were washed, and then DAB chromogenic solution was added dropwise to develop color. Finally, hematoxylin was used to stain the slides for 3 min. The slides were rinsed with running water, and then dehydrated and mounted.