Anti ap 1

Lung tissues were homogenized with a tissue lysis/extraction reagent (Sigma-Aldrich, Carlsbad, CA, United States). The concentration of proteins in each sample was determined using Bradford reagent (Bio-Rad Laboratories, Hercules, CA, United States). Equal amounts of cellular proteins (30 μg) were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated in blocking solution (5% skim milk, Millipore Co., Bedford, MA, United States), followed by overnight incubation at 4°C with the appropriate primary antibodies, as follows: anti-p65 (Cell Signaling, Denver, MA, United States), anti-p-p65 (Cell Signaling) anti-MUC5AC (Abcam), anti-AP-1 (Cell Signaling) and anti-β-actin (Cell Signaling) antibodies. The blots were washed with Tris-buffered saline containing Tween 20 (TBST), and then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson Immuno Research, West Grove, PA, United States) for 30 min at room temperature. The blots were washed again with TBST, and then, developed using an enhanced chemiluminescence kit (Thermo Scientific, San Diego, CA, United States). We evaluated the band expression values by ChemiDoc^TM (Bio-Rad, Hercules, CA, United States).

Following sacrifice, the rat left hind limbs were dissected and preserved in 10% formalin for histological assessment. As described previously [17 (link)], the sections were then deparaffinized, mounted, and stained with hematoxylin-eosin for visualization under an inverted microscope equipped with digital cameras (Olympus photomicroscope, Tokyo, Japan) for arthritis scoring in terms of cellular infiltration, joint space, pannus formation, and bone erosion. For immunohistochemical studies as described previously [18 (link)], sections were further immunostained with primary anti-NF-κB-p65 (Cell Signaling Technology, Beverly, MA, USA) and anti-AP-1(Cell Signaling Technology, Beverly, MA, USA) antibodies. Immunohistochemical scoring was carried out for NF-κB-p65 and AP-1 separately on the basis of a semi-quantitative scale and finally combined into a total score. Images were photographed randomly, and staining intensity was evaluated by an experienced pathologist on a scale of 1–4 (0 = absent, 1 = weak, 2 = moderate, 3 = high, and 4 = very high) in a blinded fashion. The sum of the scores of three independent sections of the knee joint per rat from each experimental group was averaged and plotted on a bar group.

Cells were harvested and lysed by RIPA buffer (Solarbio, Beijing, China) containing 1% PMSF protease inhibitors and phosphatase inhibitors. Equal amounts of protein (60 μg) were electrophoresed on 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and electrotransferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% bovine serum albumin for 2 h and then were incubated with primary antibodies at 4 °C overnight. The primary antibodies for CXCR2, VEGF-A, FOXD1, and HIF-1α were purchased from Abcam, and anti-FLAG, anti-JNK, anti-p-JNK, anti-ERK, anti-p-ERK anti-AKT, anti-p-AKT, anti-STAT3, anti-p-STAT3, anti-P65, anti-p-P65, as well as anti-AP-1 were purchased from Cell Signaling Technology. Then, the membranes were washed three times and incubated with secondary antibodies for 1 h at room temperature. Band intensities were quantitated by an enhanced chemiluminescence detection system (Amersham Bioscience, Piscataway, NJ, USA) according to the manufacturer’s protocol.