Beta actin antibody hrp

(E)-1-methyl-3-(phenylimino)indolin-2-one (Ligand) and (E)-1-methyl-3-(phenylimino)indolin-2-one copper(II) chloride complex (Complex) were synthesized and characterized. Dimethyl sulfoxide was from Sigma-Aldrich. Primers and Taqman probes were from Lytech (Russia). Antibodies used were anti-p53 antibody [DO-1] (ab1101, Abcam), anti-mouse IgG–Peroxidase antibody (A4416, Sigma Aldrich), THE Beta Actin antibody [HRP] (A00730, GenScript).

Total protein was extracted from BM-MSCs, PBMCs, and SH-SY5Y cells using RIPA Lysis Buffer (Thermo Fisher Scientific, San Jose, CA, USA) and a centrifugation series. Then, 15 μg of total protein was prepared for each sample, separated in 4–12% acrylamide gels, and transferred onto PVDF membranes (Bio-rad, San Jose, CA, USA). PVDF membranes were incubated in 5% milk–TBST (Tris Buffer Solution, 0.1% Tween-20, Sigma-Aldrich, St. Louis, MO, USA), incubated with primary antibodies in 5% milk–TBST, and then washed in TBST. After incubation with primary antibodies, the blots were incubated with appropriate HRP-conjugated secondary antibodies (#ab205719, Abcam, Fremont, CA, USA). Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Bio-Rad, San Jose, CA, USA). Chemiluminescence was detected with a ChemiDoc XRS⁺ system; images were acquired with the ImageLab software v6.1 (Bio-Rad, San Jose, CA, USA). The primary antibodies used in this study were: anti-Caspase-3 antibody (#ab4051, Abcam, Fremont, CA, USA), anti-Bcl2 antibody (#ab59348, Abcam, Fremont, CA, USA), Anti-BAX antibody (#ab104156, Abcam, Fremont, CA, USA), Anti-Caveolin-1 antibody (#ab2910, Abcam, Fremont, CA, USA), and THE™ beta Actin Antibody (HRP) (#A00730, GenScript, Redmond, WA, USA). Dilution was performed according to manufacturer instructions.

Cells were seeded in six-well plates at 5 × 10⁵ cells per well and cultured for 2 days followed by treatment with Complex (50 µM), Ligand (50 µM), DMSO (1%) for 24 h. Treated cells were harvested and lysed in RIPA buffer (Thermo Fischer Scientific, USA) containing 1 μl/ml Halt Protease and Phosphatase Inhibitor Cocktail with EDTA (Thermo Fischer Scientific, USA) according to manufacturer’s protocol. Whole-cell extracts were analyzed using Pierce BCA Protein Assay Kit (Thermo Fischer Scientific, USA) to determine total protein concentration. Samples were fractioned by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to Immun-Blot polyvinylidene difluoride membrane using Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad, USA). Membranes were blocked with phosphate-buffered saline (PBS) with tween 20 containing 5% (mass/vol) nonfat dried milk for 1 h at RT, incubated with primary anti-p53 antibodies (Abcam, USA) overnight at 4 °C, and then with Anti-Mouse IgG–Peroxidase antibody (Sigma-Aldrich, USA) for 1 h. THE Beta Actin Antibody [HRP] (GenScript, USA) was used for detection of Beta Actin as loading control. Blots were developed with Clarity Western ECL Substrate (Bio-Rad, USA) and documented using ChemiDoc XRS Plus (Bio-Rad, USA).