Rabbit anti gapdh monoclonal antibody

Carbon tetrachloride (CCl₄), quercetin, dimethyl sulfoxide (DMSO), olive oil, 1,4-diazabicyclo[2.2.2]octane (DABCO), and lipopolysaccharide (LPS; from Escherichia coli 0727: B8) were purchased from Sigma Chemical, Co., Ltd. (St. Louis, MO, United States). For in vivo and in vitro experiments, quercetin was diluted immediately in DMSO solution before administration.
Antibodies used in this study comprised: mouse anti-desmin monoclonal antibody (DakoCytomation, Glostrup, Denmark); rabbit anti-collagen III polyclonal antibody, rabbit anti-collagen IV polyclonal antibody, rabbit anti-CD68 monoclonal antibody, rat anti-F4/80 monoclonal antibody, mouse anti-CD11c polyclonal antibody, mouse anti-IRF5 monoclonal antibody, rabbit anti-Ym-1 monoclonal antibody, rabbit anti-CD163 monoclonal antibody, and rabbit anti-GAPDH monoclonal antibody (Abcam, Cambridge, MA, United States); rabbit anti-IL12a monoclonal antibody, rabbit anti-Notch1 monoclonal antibody, and rabbit anti-β-actin monoclonal antibody (Cell Signaling Technology, Boston, MA, United States).

Control (20 nM) or miR-4669 mimic (10 or 20 nM)-transfected Hep3B cells were cultured for 72h, and GAPDH levels in the culture medium were semi-quantified using western blot analysis. Briefly, proteins in culture media (10 μL) were fractionated and transferred onto a PVDF membrane (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA). The membrane was immunoprobed with a rabbit anti-GAPDH monoclonal antibody (×500 dilution, Abcam, Cambridge, UK) followed by incubation with HRP-conjugated anti-rabbit IgG (×5000 dilution, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Signals were visualized using an Immunobilon Forte Western HRP substrate (MilliporeSigma), and signal intensity was quantified using a G:BOX Image Station iChemi XL device (SYNGENE, Cambridge, UK).

Total protein (50 μg) extracted from liver tissues or hepatic macrophages was used for Western blot analysis. The following primary antibodies were used: rabbit anti-iNOS monoclonal antibody (1:1000, Abcam), rabbit anti-GAPDH monoclonal antibody (1:10000, Abcam) or Mouse anti-Actin monoclonal antibody (1:10000, Abcam). Goat anti-mouse IgG labeled with HRP (Cat. No. ab6789, 1:2000, Abcam) was used as the secondary antibody. Immunoreactive signals were detected using an Enhanced Chemiluminescence (ECL) kit (Amersham Pharmacia Biotech) through an ECL system. The results were quantified using Image J 1.43 (National Institutes of Health, Bethesda, MD) after densitometric scanning of the films. Western blot signals were normalized relative to the image of the appropriate control samples. Results of a minimum of three independent Western blot analyses were averaged and pooled to yield the data shown in the histograms.

Western blotting was performed according to our previous publication (9 (link), 10 (link)). The antibodies used included rabbit anti-Gankyrin monoclonal antibody (1:1000; Abcam, USA), rabbit anti-GAPDH monoclonal antibody (1:1000; Abcam, USA) and goat anti-rabbit IgG-horse radish peroxidase (HRP)-linked secondary antibody (1:1500; Thermo Fisher Scientific, Inc. USA). Briefly, after blocking with 5% non-fat milk dissolved in Tris-buffered saline containing Tween-20 (TBST) at 37°C for 1 hour, the membranes were incubated with the anti-Gankyrin or anti-GAPDH primary antibody at 4°C overnight. The following day, the membranes were incubated with the secondary antibody at room temperature for 1 hour. The specific bands were developed using an enhanced chemiluminescence reagent in a gel imaging system, and the bands were analyzed using Image J software. A total of three independent experiments were performed.

Canine LAA tissues were lysed with RIPA Lysis Buffer (ASPEN, USA) supplemented with complete protease and phosphatase inhibitor cocktail (ASPEN, USA). We used Bicinchoninic acid (BCA) assay (ASPEN, USA) to estimated protein concentration after centrifugation at 13000 rpm for 5 min. Proteins were separated on SDS-polyacrylamide gels and transferred to PVDF membranes. Then, the membranes were incubated overnight at 4 °C with the following primary antibodies: rabbit monoclonal anti-GAPDH antibody (diluted 1:10000, Abcam), rabbit monoclonal anti- p-AMPKα1 antibody (diluted 1:1000, Abcam), rabbit monoclonal anti- PGC-1α antibody (diluted 1:500, Abcam), rabbit monoclonal anti-PPARα antibody (diluted 1:1500, Abcam), rabbit monoclonal anti-FAT antibody (diluted 1:500, Bioss), rabbit monoclonal anti-VLCAD antibody (diluted 1:1000, Abcam) and then with secondary HRP-goat anti-rabbit antibody (diluted 1:10000, ASPEN) for 30 min at room temperature (RT). For loading controls, the membranes were stripped with stripping buffer (ASPEN, USA) for 10 min at room temperature. Antibody binding was detected with the ECL detection reagent (ASPEN, USA). At last, we quantified bands with AlphaEaseFC Software and data are shown as the ratio of total protein to GAPDH normalized to control.