Horseradish peroxidase conjugated goat anti rabbit secondary antibody

Protein was extracted from LX-2 cells, NK-92 cells, or liver tissues with RIPA protein lysis buffer (1% Triton X-100, 1% deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) (Beyotime, Shanghai, China). The protein concentration of each sample was determined by the BCA assay. Proteins (30 μg/lane) were separated by SDS-polyacrylamide gel electrophoresis using a 10% polyacrylamide gel, and then transferred to PVDF membranes (Millipore, Billerica, MA, United States). The following primary antibodies were used: Anti-phosphorylated extracellular signal-related kinase (p-ERK) (#14227S; Cell Signaling Technology, Danvers, MA, USA), anti-ERK (#4348S, Cell signaling Technology), alpha smooth muscle actin (α-SMA) (#4668S; Cell signaling Technology), Collagen I (WL008; Wanleibio Co., Ltd., Shenyang, China), and β-actin (#3700; Cell signaling Technology). For ECL detection, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (#A0208; Beyotime, Shanghai, China) at a dilution of 1:10000 for 1 h at 37ºC. ECL detection was performed according to the manufacturer’s protocol (Millipore). Analysis of blots was performed using Image J.

Proteins from liver tissues were isolated with RIPA buffer (Beyotime, Haimen, China) and
were quantified with a bicinchoninic acid (BCA) protein quantitation kit (Beyotime). Equal
amounts of proteins were separated by 5% or 10% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and electrophoretically transferred onto polyvinylidene
difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After blocking with 5% nonfat
milk, the membranes were incubated with primary antibodies (rabbit anti-CYP2E1 antibody,
rabbit anti-CYP1A2 antibody, rabbit anti-p65 antibody, each 1: 1,000 diluted, purchased
from proteintech, Wuhan, China; rabbit anti-p-p65^Ser536 antibody, rabbit
anti-p-IkB^Ser32 antibody, rabbit anti-IkB antibody, each 1: 1,000 diluted,
purchased from Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. After that,
the membranes were incubated with a horseradish peroxidase-conjugated goat anti-rabbit
secondary antibody (1:5,000 dilution, Beyotime) at 37°C for 45 min. Finally, the specific
protein bands were visualized with an enhanced chemiluminescent (ECL) reagent
(Beyotime).

Cytoplasmic and nuclear proteins were isolated from lung tissue samples following the instructions of the KGI Nuclear and Cytoplasmic Protein Extraction kit (Nanjing KGI Biotechnology). Protein concentrations were determined using the BCA method (Beyotime Institute of Biotechnology, Haimen, China). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and were transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA). The membranes were incubated overnight at 4°C with rabbit polyclonal antibodies against TLR4, NF-κB p65, Nrf2, HO-1, and activated caspase-3 (Santa Cruz Biotechnology, Inc., Dallas, TX). The membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Beyotime Institute of Biotechnology) at 37°C for 45 min. The enhanced chemiluminescence (ECL) imaging system was to visualize protein bands. β-actin was used as the internal control.

Total protein and nucleoprotein were extracted from the middle lobe of the right lung using the Thermo protein extraction kit (Thermo, Waltham, MA, USA). Protein concentrations were determined using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology, Haimen, China). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were incubated overnight at 4°C with rabbit polyclonal antibodies against total Akt, p-Akt (Cell Signaling Technology, Inc. Danvers, MA, USA), Nrf2 (Biotechnology Inc, Dallas, TX, USA), HO-1 and activated caspase-3 (Santa Cruz Biotechnology Inc, Dallas, TX, USA). The membranes were then incubated with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Beyotime, Jiangsu, China) at 37°C for 45 min. An enhanced chemiluminescence (ECL) imaging system was used to visualize the protein bands. The Akt phosphorylation level was determined by the ratio of p-Akt to total Akt expression.

To assess the levels of TGF-β₁ protein in nasal and lung tissues, IHC was performed as described previously (21 (link)). Briefly, nasal mucosa and lung sections were incubated with mouse monoclonal anti-TGF-β₁ antibody (1:20; rabbit monoclonal antibody; ab170874; Abcam) at 4°C overnight, followed by incubation with horseradish peroxidase conjugated goat anti-rabbit secondary antibody (1:200; A0208; Beyotime Institute of Biotechnology, Haimen, China) at 37°C for 2 h. Sections were then developed with diaminobenzidine (DAB) in accordance with instructions of a commercial DAB Horseradish Peroxidase Color Development kit (P0202; Beyotime Institute of Biotechnology). Positive stained cells were counted under a light microscope at ×200 magnification.

Target proteins were extracted from MC3T3-E1 cells using RIPA lysis buffer (Jrdun Biotechnology) and the protein concentration was determined by a bicinchoninic acid assay kit (Thermo Fisher Scientific, Inc.). The isolated proteins (25 µg/lane) were separated by electrophoresis in 10% SDS-polyacrylamide gels, and transferred onto a PVDF membrane. Membranes were subsequently blocked with 5% non-fat milk overnight at 4˚C, and incubated with the following primary antibodies: Anti-PPARγ2 (1:500; cat. no. ab45036; Abcam), anti-bone morphogenetic protein-2 (BMP2; 1:1,000; cat. no. orb334018; Biorbyt, Ltd.), anti-runt-related protein 2 (Runx2; 1:1,000; cat. no. ab23981; Abcam), anti-osteocalcin (OCN; 1:1,000; cat. no. ab93876; Abcam) and anti-GAPDH (1:2,000; cat. no. 5174; CST Biological Reagents Co., Ltd.) overnight at 4˚C. Following primary incubation, membranes were incubated with the horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Beyotime Institute of Biotechnology; cat. nos. A0208 and A0216; both 1:1,000) at 37˚C for 1 h. Signal quantification was performed by an enhanced chemiluminescence system (Bio-Rad Laboratories, Inc.). The bands were quantified by densitometry with ImageJ software (version 1.51; National Institutes of Health).