Goat anti rabbit igg hrp antibody

Allele specific gene expression analysis was conducted with data from RNA sequencing (Macrogen). For cell cycle analysis, LCLs were fixed with 70% ethanol for 30 min at 4°C. Cells were then stained with 40 μg/mL propidium iodide (Sigma-Aldrich) and 100 μg/mL RNase A by incubating in dark at room temperature for 1 hr. Stained cells were analyzed by flow cytometry. RAD21 Western blot was conducted with anti-RAD21 antibody (1 : 750, ab42522, Abcam) and goat anti-rabbit IgG-HRP antibody (1 : 2000, sc-2004, Santa Cruz). GAPDH (1 : 2500, G9295, Sigma-Aldrich) was used as a loading control. For chromosome counting, 5 × 10⁵ LCLs were cultured in 2 mL medium and treated with 100 ng/mL colcemid (KaryoMAX, Gibco) for 1 hr. Cells were incubated with 50 mM KCl at 37°C for 15 min. Cells were fixed with 10 mL cold fixation solution (methanol and acetic acid (3 : 1 v/v)) and stored at -20°C. Cells were resuspended in freshly made fixation solution and dropped on HistoBond+ slides (Marienfeld Superior). Slides were then stained with Giemsa and chromosomes were counted.

7, 8-dihydroxyflavone (TCI laboratories, Tokyo, Japan) was dissolved in dimethylsulfoxide (DMSO)to 100 mM as a stock solution. ANA-12 (Cat^# BTB06525, N2-(2-phenyl)benzo-thiophene-2-carboxamide) was purchased from Maybridge (Fisher Scientific Worldwide, Shanghai, China); H₂O₂ (hydrogen peroxide)and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) from Sigma-Aldrich (St. Louis, MO, USA); DAPI from Immunochemistry Technologies (Bloomington, USA); Antibodies against cytochrome C, Akt, phosphorylated Akt, NF-κB and IκB were purchased from Cell Signaling Technology (Danvers, MA, USA); antibody against HO-1 from Abcam (Cambridge, MA, USA); the anti-β-actin antibody was procured from Bioss (Beijing, China); goat anti-rabbit IgG-HRP antibody from Santa Cruz Biotechnology (Paso Robles, CA, USA);Goat anti-rabbit IgG antibody conjugated with FITC from Sigma-Aldrich (St. Louis, MO, USA).

Exosomal EV preparations (1 μg protein, as quantified by the Bradford assay) were denatured in Laemmli buffer and by boiling for 5 min. For better resolution of proteins on SDS polyacrylamide gels, the following acrylamide concentrations were used: 10% for Grp-78, 12% for CD63 and 15% for sPLA₂-IIA. Following transfer onto PVDF membranes (Laemmli, 1970), they were incubated with blocking solution (5% non-fat milk in TBS-Tween 20) overnight at 4°C and then incubated with the appropriate dilutions of primary antibodies in PBS (overnight at 4 °C) as follows: CD63 (E-12) (sc-365604, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA, mouse monoclonal, dilution 1:1000), sPLA₂ group IIA (ab23705, ABCAM, Cambridge, MA, USA, polyclonal rabbit anti-human, dilution 1:1000) and Grp-78 (H-1296) (sc-13968, Santa Cruz Biotechnology, Inc., CA, USA, dilution 1:1000). After washing with TBS-T, blots were incubated for 2 h at room temperature with the appropriate secondary antibodies: goat anti-rabbit IgG-HRP antibody (sc-2004, Santa Cruz Biotechnology, Santa Cruz, CA, USA, dilution 1:10,000 in TBS-Tween 20) and goat anti-mouse IgG-HRP, (sc-2005, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA, dilution 1:10,000 in TBS-Tween 20). The immunoreactive bands were visualized with Luminata Crescendo Western HRP Substrate, WBLURO100 (Millipore, Temecula, CA, USA).

Total protein was isolated from PBMCs by using RIPA buffer (Abcam, Cambridge, United Kingdom). The amount of proteins was quantified by BCA Protein assay kit (Thermo Fisher Scientific, Rockford, IL, United States), with bovine serum albumin (BSA) as a standard. Each protein sample was loaded onto 10% SDS-polyacrylamide gel and transferred to nitrocellulose membrane (Schleicher & Schuell BioScience, Germany) for 90 min at 4°C, and blocked with 5% skim milk in TBST (1 M Tris–HCl, 5 M NaCl, 10% Tween-20) for 1 h at room temperature. The blots were incubated with anti-GATA3, -T-bet, -C-maf, -STAT1, -P-STAT1, -STAT4, -P-STAT4 or -beta-actin (all from Abcam) antibodies overnight at 4°C. Primary antibody-bound membranes were incubated with goat anti-rabbit IgG-HRP antibody (Santa Cruz Biotechnology, United States) for 1 h at room temperature. The target protein was visualized with enhanced chemiluminescence system (GE Healthcare Life Sciences, United States), followed by analysis using ChemiDoc XRS (Bio-Rad).