Hrp labeled goat anti rabbit igg

Cells were lysed in cell lysis buffer (Beyotime Biotechnology, China) containing 1 mM phenylmethylsulfonyl fluoride (PMSF). Total protein concentrations in the supernatant were measured with a bicinchoninic acid assay (BCA) (Beyotime biotechnology, China), and protein (30 μg/lane) was separated on 10% polyacrylamide gels (Servicebio, Wuhan, China) with electrophoresis and subsequently transferred onto PVDF membranes. Membranes were blocked with 5% skim milk in TBST at room temperature for 1 h. The membranes were incubated with primary antibodies against Slug (Cell Signaling Technology, 1:2000), LAMB3 (Abcam, 1:1000), Podoplanin/gp36[EPR22182] (Abcam, 1:2000), E-cadherin (Cell Signaling Technology, 1:1000), and Vimentin (D21H3) XP® (Cell Signaling Technology, 1:1000) overnight at 4 °C. After washing three times for 5 min in TBST, the membranes were incubated for 1 h at 37 °C with an HRP-labeled goat anti-rabbit IgG (Proteintech, Wuhan, China) diluted 1:5000 in TBST. Bands were visualized using a WesternBright Sirius Chemiluminescent Detection Kit (Advansta, California, USA). GAPDH protein levels were used as loading controls. The experiments were repeated three times.

Immunohistochemistry was performed using an indirect peroxidase method. Paraffin embedded sections of esophageal tissues on slides were dewaxed fully in xylene and rehydrated thoroughly in a decreasing graded series of ethanol concentrations. Endogenous peroxidase was quenched with 3% hydrogen peroxide, and sections were blocked with 10% goat serum (ZSGB-BIO; Beijing, China) to reduce non-specific binding of antibodies. All tissues were incubated overnight with primary antibody at 4 °C. Antibody against TGFBR2 was obtained from Abcam (Shanghai, China). For detection, slides were returned to room temperature and incubated with horse radish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:200, Proteintech, Wuhan, China). Diaminobenzidine (DAB, ZSGB-BIO, Beijing, China) was used as the chromogenic substrate. Slides were counterstained with hematoxylin and mounted in resin. Images were acquired through Aperio pathology scanner.

The cells were lysed in cell lysis buffer (Beyotime Biotechnology, China) to obtain the protein, and the concentration of the protein was measured with a BCA Kit (Beyotime Biotechnology, China). Protein (30 μg per lane) was added to a polyacrylamide gel and transferred to a PVDF membrane after separation by electrophoresis. Subsequently, the membrane was blocked with TBST diluted 5% skim milk for 1 hr at room temperature. Primary antibodies against ATAD2 (1:1000; Cell Signaling Technology), E-cadherin (1:1000; Cell Signaling Technology), N-cadherin (1:1000; Cell Signaling Technology) and Snail (1:1000; Cell Signaling Technology) were used for binding protein specifically at 4°C overnight. After washing three times for 10 min in TBST, membranes were incubated with an HRP-labeled goat anti-rabbit IgG (1:10000, Proteintech, Wuhan, China) for 1 hr at 37°C. The WesternBright Sirius Chemiluminescent Detection Kit was used for the visualization of bands on PVDF membranes. GAPDH (1:10000, Bioprimacy) was used as an internal reference protein. The experiments were repeated three times.

The influenza A virus H1N1 strain (A/PR8/34)-adapted mouse lung was kindly presented by the College of Life Sciences, Hunan Normal University (Changsha, China). It was cultured in 9-day-old chicken embryo and the allantoic fluid was harvested at 3 days after culture, filtered using a 0.22 μm filter, then stored at −80 °C. The H1N1 virus titer was determined by a blood coagulation assay. The Madin–Darby canine kidney (MDCK) cell line was purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China). In total, 0.25% EDTA-trypsin, fetal bovine serum, and Trizol were purchased from Gibco (Thermo Fisher, Waltham, MA, USA). DMEM high-glucose culture medium (containing sodium pyruvate) was purchased from Hyclone (Logan, UT, USA). TPCK–trypsin was purchased from Sigma Chemical Co. (St. Louis, MO, USA). RIPA lysate was purchased from Well Biological Science Co. (Changsha, China). Rabbit anti-dog NF-κB, TLR4, MyD88, mouse anti-dog β-actin, HRP labeled goat anti-rabbit IgG, and goat anti-mouse IgG antibodies were purchased from Proteintech Group, Inc (Chicago, IL, USA). Quantitative real-time polymerase chain reaction (QRT-PCR) primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China), and the mRNA reverse transcription kit and Ultra-SYBR Mixtures were purchased from CoWin Bioscience Co., Ltd. (Changsha, China).

Selenium powder, sodium borohydride, isophorone diisocyanate (IPDI), methoxypolyethylene glycol (mPEG), dibutyltin dilaurate (DBTDL), 11-bromoundecanol, methoxypolyethylene glycols, Tetrahydrofuran (THF), hydrogen peroxide (H₂O₂), Cy 5 (Aladdin, Shanghai, China). Isoginkgetin (IGK), chloroquine (CQ), (MedChemExpress, Monmouth Junction, USA). Primary antibodies against aggrecan, collagen II, SOX9, MMP3, MMP13, ADAMTS5, ATG7, Beclin1, LC3B, SQSTM1/p62 (Abcam, Cambridge, UK), primary antibodies against BCL2, BAX and cleaved caspase-3 (Cell Signalling Technology, Danvers, USA), primary antibodies against β-actin (Proteintech, Wuhan, China). Goat anti-rabbit IgG H&L (Alexa Fluor^® 488), goat anti-rabbit IgG H&L (Alexa Fluor^® 594), (Abcam, Cambridge, UK), HRP-labeled goat anti-rabbit IgG (Proteintech, Wuhan, China). Penicillin–streptomycin (Sigma-Aldrich, St. Louis, USA). Cell counting kit-8 (CCK-8), Calcein/PI assay kit, ROS assay kit, and TUNEL apoptosis assay kit (Beyotime, Shanghai, China). Phosphate-buffered saline (PBS), FITC-annexin V/PI apoptosis detection kit, fetal bovine serum (FBS), DMEM/F12 (Thermo Fisher Scientific, Waltham, USA). Nitrocellulose membranes (Pall, New York, USA). Electrochemiluminescence substrate (Meilunbio, Dalian, China). Zoletile (Virvac, Carros, France).