Horseradish peroxidase hrp conjugated goat anti rabbit igg

The purified proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Silver Staining, and Western blot. Briefly, after the supernatants were boiled in Laemmli sample buffer containing 2% SDS with beta- mercaptoethanol (β-ME), the proteins were separated by Tris-Glycine SDS-PAGE gels and transferred to nitrocellulose membrane. After blocking for 1 h at room temperature (RT) with 5% non-fat milk in TBS-T, rabbit anti-SARS-CoV spike polyclonal antibody (1:3000) (Sino Biological), or rabbit anti-SARS-CoV nucleoprotein (1:3000) (Sino Biological) was added and incubated overnight at 4 °C as primary antibody, and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:10,000) (Jackson immuno research) was added and incubated at RT for 1 hs as secondary antibody. After washing, the signals were visualized using ECL Western blot substrate reagents and iBright 1500 (Thermo Fisher).

Cells were plated at 6×10⁵ cells/well in six-well plates and transfected as described above.
The proteins were extracted, and the total protein concentration was determined by Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA, USA) following manufacturer’s manual. The absorbance was measured by a spectrophotometer (CT-5600; ChromTech, Inc, Apple Valley, MN, USA). Proteins were separated in 8%–13.5% polyacrylamide-SDS gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% skim milk in TBST (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween 20), the membranes were incubated with specific antibodies against Calnexin, HuR, HA, c-Myc, caspase-3, -9 (Cell Signaling Technology Inc., Beverly, MA, USA), galectin-3, P-gp, MRP1, Bcl-2 (GeneTex Inc., Hsinchu, Taiwan), and β-actin (Millipore, Billerica, MA, USA) overnight at 4°C. These membranes were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Inc., PA, USA) for 1 h at room temperature. The detection of the signal was performed by incubating blotted membranes with enhanced chemiluminescence detection kit (PerkinElmer Life Sciences, Waltham, MA, USA).

hMSCs were provided from the Medipost, Co. (Seoul, Korea). Fetal bovine serum was purchased from Bio Whittacker (Walkersville, MD, USA). The following antibodies were purchased: F-actin, phospho-PKC, phospho-DRP1, Gα_q, Gα_i and Gα₁₂ antibodies (Cell Signaling Technology, Danvers, MA, USA); Cdc42, Rac1 and RhoA antibodies (BD Biosciences, Franklin Lakes, NJ, USA); DRP1, OPA1, Fis1, MFN2, p-ERK, p-JNK, ERK, JNK, GPR91, PKCδ, PKCε, PKCθ, PKCζ, profilin-1, phospho-cofilin1, cofilin1, β-actin and and β-tubulin antibodies (Santa Cruz Biotechnology, Paso Robles, CA, USC); COX IV antibody (Abcam, Cambridge, England); MFN2 antibody (Proteintech, Wuhan, China); Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Jackson Immunoresearch, West Grove, PA, USA). Succinate, NAC and mitomycin C were obtained from Sigma Chemical Company (St. Louis, MO, USA).

Fetal bovine serum (FBS), trypsin–EDTA and penicillin–streptomycin were obtained from BioWhittaker (San Diego, CA, USA). 3-(4,5-Dimetylthiazol-yl)-diphenyl tetrazolium bromide (MTT) was purchased from DUCHEFA Biochemie (Haarlem, Netherlands). Dulbecco’s modified eagle’s media (DMEM), Nutrient Mixture F-12 Ham medium, mannitol and d-glucose, were supplied by Sigma Chemical (St. Louis, MO, USA), as were all other reagents unless specifically stated otherwise. AGE-BSA antibody was provided by Bioss Antibodies (Woburn, MA, USA). FSP-1 antibody was obtained from AbCam (Cambridge, UK). Antibodies of α-SMA, collagen 1, collagen IV, RAGE, MMP-2, MMP-9 and CTGF were provided by Santa Cruz Biotechnology (Dallas, TX, USA). MT-1 MMP antibody was purchased from R&D system (Minneapolis, MN, USA). Antibodies of TGF-β receptor I (RI), TGF-β receptor II (RII) and Smad 2/3 were obtained from Cell Signaling Technology (Beverly, CA, USA). TGF-β RI kinase inhibitor was obtained from Calbiochem (Darmstadt, Germany). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, goat anti-mouse and donkey anti-goat IgG were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).
Chrysin (Sigma-Aldrich Chemical) was dissolved in dimethyl sulfoxide (DMSO) for live culture with cells; a final culture concentration of DMSO was <0.5%.

Extracted protein was boiled in SDS/β-mercaptoethanol sample buffer, and 50 μg protein was run on 5–12% gradient polyacrylamide gel and transferred to PVDF membranes (Amersham, St Albans, Herts, UK). Membranes were incubated with rabbit anti-MECP2 polyclonal antibody (GeneTex, USA, 1:1000), mouse anti-β-ACTIN monoclonal antibody (Abcam, Cambridge, MA USA, 1:1000), rabbit anti-DNMT1, DNMT3a and DNMT3b polyclonal antibody (1:1000, SantCruz Biotechnology Inc., CA, USA) overnight at 4 °C. After that, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Jackson Immunoresearch Laboratories, Inc., West Grove, PA, USA, 1:10, 000) at room temperature for 1 h. ECL detection reagents (Millipore, Billerica, MA, USA) were added on the membranes for 1 min and were immediately exposed to X-ray film (Kodak, USA). The β-actin signal was used as a loading control. The experiment has been repeated at least three times. The bands were analyzed using Quantity One analyzing system (Bio-Rad, Hercules, CA, USA). The protein level was represented as the relative ratio of the MECP2, DNMT1, DNMT3a and DNMT3b signals vs the housekeeping gene (β-ACTIN).