Horseradish peroxidase hrp

Frozen brachiocephalic artery sections were stained with antibodies against CD68 (marker for macrophages, MCA1957, AbD Serotec, UK), ADAMTS4 (a generous gift from Prof. Hideaki Nagase, Kennedy Institute of Rheumatology, Oxford University, UK), alpha smooth muscle actin (αSMA) (CGA7, Santa Cruz Biotechnology Inc., USA), ADAMTS4/1 cleaved versican V1 neoepitope DPEAEE (ab19345, Abcam, USA) or aggrecan ARGxx neoepitope (ab3773, Abcam, USA) with the corresponding secondary antibody conjugated to horseradish peroxidase (HRP) (Santa Cruz Biotechnology Inc., USA) and visualized by applying 3,3′-diaminobenzidine tetrahydrochloride (DAB) or through Alexa fluor 488 or 568 (Life technologies, Singapore) and visualized under Axiovision compound microscope (Zeiss, Germany) or UltraView Vox Spinning Disk confocal microscopy (PerkinElmer, USA). Percentage positive staining was quantified using Adobe Photoshop CS8 software (USA). The vulnerability index was calculated as the ratio of (ORO stained area + CD68 positive area)/(αSMA area + collagen positive area). The specificity of the antibodies used is demonstrated using isotype antibody controls in all IHC/IF staining (Supplementary Fig. S9).

Resveratrol, D-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS) was obtained from Aladdin Chemicals (Shanghai, China). Stearic acid, lecithin chloroform, and Tween80 were acquired from Sinopharm Chemical Reagent Co, Ltd. (Shanghai, China). Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin G, and trypsin-EDTA were purchased from Thermo Fisher Scientific (MA, United States). Other chemicals used in this study were of analytical grade. The primary antibodies used in the experiment including P-gp, BCRP, N-cadherin, Vimentin, MMP-2, and MMP-9 were purchased from Proteintech (Beverly, MA, United States). Horseradish peroxidase (HRP) which are conjugated secondary antibodies used against rabbit or mouse immunoglobulin were purchased from Santa Cruz Biotechnology (Santa Cruz).

Mouse monoclonal antibodies (STAT3, STAT5, ERK1/2, Akt, NF-κB, IL-6, actin, p-ERK1/2, and p-p38), rabbit polyclonal antibodies (cleaved IL-1β, TLR-2, TLR-4, TLR-5, p38, p-STAT3, p-STAT5, p-Akt1, and p-NF-κB), and goat polyclonal antibodies (TNF-α and IL-1β) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The samples derived from cells and lung homogenates were lysed in radioimmunoprecipitation assay (RIPA) buffer, separated by electrophoresis on SDS-PAGE gels, and transferred to nitrocellulose transfer membranes (GE Amersham Biosciences, Pittsburgh, PA). Proteins were detected by Western blotting using primary Abs at a concentration of 1/200 and were incubated overnight (51 (link)). Labeling of the first Abs was performed using relevant secondary Abs conjugated to horseradish peroxidase (HRP) (Santa Cruz Biotechnology), which were detected using ECL regents (Santa Cruz Biotechnology) and quantified using Quantity One software (Bio-Rad).

An anti-FAM83G antibody was produced by the Dundee Division of Signal Transduction and Therapy (DSTT) as a sheep polyclonal antibody against the C-terminus of FAM83G (S876C); sheep anti-GFP was also produced by the DSTT (S268B). Other antibodies used in these studies were: Myc tag (cat. no. CST2276; Cell Signaling Technology); FLAG-M2-HRP (cat. no. A8592; Sigma); MYC-HRP (cat. no. 11814150001; Roche); CD2AP (gift from Andrey Shaw, Research Biology, Genentech, South San Francisco, CA), CD2AP (clone 2A2.1, cat. no . MABT419; Millipore), anti-FAM83G (cat. no. HPA023940; Sigma), and actin (cat. no. ab8227; Abcam). Secondary rabbit, mouse and sheep antibodies conjugated to horseradish peroxidase (HRP) were used at 1:10,000 (Santa Cruz Biotechnology). Fluorophore-conjugated phalloidin (Alexa Fluor 488 and Atto 562) and Alexa-Fluor-594-conjugated anti-mouse-IgG (ThermoFisher) were used for fluorescence microscopy.

Ras-related C3 botulinum toxin substrate 1/Cell division control protein 42 homolog activator (#CN02-A, Cytoskeleton, Inc., San Diego, CA, United States), Antibodies used for immunoblotting and immunofluorescence were as follows: P-Cav-1 (#3251), T-Cav-1 (#3267), P-Src (#2101), T-Src (#2108), and GAPDH (#5274) were all from Cell Signaling Technology (San Diego, CA, United States). P-cofilin (sc-271921) was from Santa Cruz Biotech (Santa Cruz, CA, United States), microtubule-associated protein 2 (MAP2) (ab5392) was from Abcam (Cambridge, MA, United States), anti-neurofilament SMI 31 (#801601) was from BioLegend (San Diego, CA, United States). Primary antibodies were visualized using secondary antibodies conjugated to horseradish peroxidase (HRP) (Santa Cruz Biotech, Santa Cruz, CA, United States) and lumigen ECL ultra (MA-100, Lumigen Inc., Southfield, MI, United States). All displayed bands were compared to molecular weight standards (sc-2035, Santa Cruz Biotech, Santa Cruz, CA, United States). The amount of protein per sample was determined using a dye-binding protein assay (Bio-Rad, Hercules, PA, United States). For immunofluorescence, FITC-488, Texas Red-595 and Cy5-647 secondary antibodies were obtained from Molecular Probes (Carlsbad, CA, United States).

Bacillus Calmette-Guerin Vaccine (BCG) frozen powder (80 mg/vial) was purchased from Beijing institute of biological products. Complete Freund’s adjuvant (10 ml/vial) and Bovine collagen type II (C-7806) were purchased from Sigma Co. USA. Tumor necrosis factor-α (TNF-α), IFN-γ, IL-4 and IL-17 ELISA kits were purchased from Shanghai Sengxiong Technological Co. China. Fluorescein isothiocyanate (FITC)-conjugated anti-rat CD4 antibody and allophycocyanin (APC)-conjugated anti-rat CD8 antibody were purchased from eBioscience USA. Anti-rat TNF-α, IFN-γ, IL-4 and IL-17, monoclonal anti-rat HIF-1α and NF-κB, and horseradish peroxidase (HRP) were purchased from Santa Cruz Biotechnology, USA. SP9001 kit and DAB (3,3’-diaminobenzidine) color reagent were purchased from Beijing Zhongshan Jinqiao Biotech Co. Ltd. China. BCA kit was purchased from Pierce Co. USA.