β actin mouse monoclonal igg1

Immunofluorescence staining was carried out to further validate the effect of FA-g-CS/ANC on the expression of sGLT1 and GLUT2. In short, Caco-2 cells were transfected with NC siRNA (negative control), GLUT2-siRNA-2, and sGLT1-siRNA-3; then, they were seeded onto a 4-well slide and chamber (Watson, Kobe, Japan). Subsequently, cells were incubated with 0.09 µg/mL FA-g-CS, 0.23 µg/mL ANC, or their combination at 37 °C for 48 h. Then, they were fixed with 4% paraformaldehyde for 10 min and soaked with 0.5% Triton X-100 in PBS for 15 min. In this study, goat serum was used to avoid nonspecific binding at room temperature. After 30 min of treatments, the cells were incubated with the primary antibody (β-actin mouse monoclonal (IgG1), Santa Cruz Biotechnology, with 1:100 dilutions) at 4 °C overnight [31 (link)], followed by incubation with the secondary antibody (goat anti-mouse IgG1-HRP, Santa Cruz Biotechnology, Shanghai, China, with 1:100 dilutions) for 1 h at 20–37 °C. Nuclear staining was performed with DAPI, and the images were captured with a fluorescence microscope (BX53, Olympus, Tokyo, Japan).

Delphinidin-3-glucoside (ANC) was purchased from Ziguang (Nanjing, China). FA-g-MD was prepared by our group. 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH) and 2′,7′-dichlorofluorescin diacetate (DCF-DA) were purchased from Aladdin Reagent (Shanghai, China). HT-29 cells were obtained from Chinese Academy of Sciences (Kunming, China). DMEM, DMEM/Nutrient Mix F12 (1:1) medium, fetal bovine serum, penicillin/streptomycin and molecular protein marker were sourced from Invitrogen GmbH (Karlsruhe, Germany). Cell culture consumable materials were purchased from Greiner Bio-One (Essen, Germany). Monoclonal antibody quinone oxidoreductase 1(NQO1), glutathione reductase (GSR), γ-glutamatecysteine ligase catalytic submit (γ-GCLC), and β-actin mouse monoclonal (IgG1) and secondary antibodies (goat anti-mouse IgG1-HRP, goat anti-rabbit IgG-HRP) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). All organic solvents and other chemicals were of analytical grade and complied with the standards needed for cell culture experiments.

Western blot analysis was performed as described by Upadhyay et al. [21 (link)]. Protein concentration was estimated in tissue homogenate using Bradford reagent (Sigma, Germany). Primary antibody COL3A1-mouse monoclonal IgG₁ (sc-271249), bFGF-mouse monoclonal IgG_2a (sc-74413), Smad 2-goat polyclonal IgG (sc-6200), Smad 4-rabbit polyclonal IgG (sc-7154), Smad 7-mouse monoclonal IgG₁ (sc-365846), β-Actin-mouse monoclonal IgG₁ (sc-47778), and respective secondary antibody goat anti-mouse IgG-AP (sc-2008), rabbit anti-goat IgG-HRP (sc-2768), and goat anti-rabbit IgG-AP (sc-2007) were purchased from Santa Cruz Biotech. (USA). Smad 3-rabbit polyclonal IgG (Cat-10832) was purchased from Cayman Chemicals (USA). Equal amount of protein was electrophoresed on 12% SDS-PAGE with 4% stacking gel (Mini Trans-Blot, BioRad Laboratories Inc., USA) at 80 V for 45 min. Proteins were transblotted onto the PVDF membrane (Millipore Corp., USA), and processed with COL3A1, bFGF, Smad-2, -3, -4, -7 and β-Actin primary antibodies (1 : 1000) and corresponding secondary antibodies (1 : 2000). The desired proteins were detected by BCIP-NBT solution and western Max-HRP-Chromogenic detection kit (Amresco, USA). β-actin was estimated as internal control.