Anti cleaved parp1

Whole cell lysates were prepared from A2780 or SKOV3 cells transfected with indicated siRNAs for 48 or 72 h. Western blotting using anti-cleaved PARP1 (Abcam, Cambridge, UK) and β-actin (Sigma–Aldrich) antibodies and subsequent detection were performed as described [46 (link)].

KG-1 cells were plated in a 6-well plate at a density of 10⁶ cells/mL before treatment with two increasing concentrations of ASEE for 24 h (38 and 76 μg/mL). Control cells were treated with RPMI media. Total proteins were extracted using the Q-proteome Mammalian Protein kit (Qiagen, Hilden, Germany) and quantified using the DC (Detergent Compatible) protein assay (Bio-Rad). Proteins were separated by SDS-PAGE; transferred to PVDF (Polyvinylidene fluoride) membranes which were blocked with 5% skimmed milk; and then incubated with primary antibodies anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), anti-Cytochrome-c, anti-cleaved PARP-1, anti-Bax, anti-Bcl2, and anti-caspase-9 (Abcam, Cambridge, UK), anti-p53, and anti-caspase-8 (Elabscience, Houston, TX, USA) at the manufacturer’s recommended concentrations. After washing and incubation with a secondary antibody (Bio-Rad, Hercules, CA, USA), membranes were washed and image development was done using the Clarity™ Western ECL Substrate (Abcam, Cambridge, UK) on the ChemiDoc machine (BioRad, Hercules, CA, USA). Blot bands were quantified using the ImageJ computer program to calculate the relative expression of proteins.

For Western blotting, skeletal muscle tissues were homogenized with RIPA buffer. After the lysates were centrifuged at 13,000 rpm for 30 min, the supernatants were measured using bicinchoninic acid (BCA) (Thermo Fisher Scientific, Waltham, MA, USA) assay and boiled for 5 min at 100 °C after mixing with a 5× sample buffer. Western blotting samples were loaded onto SDS-PAGE gels, run, and transferred onto PVDF membranes (Merck Millipore, Burlington, MA, USA). After blocking with Tris Buffered Saline with Tween 20 (TBST) containing 5% skim milk, the membranes were incubated with primary antibodies overnight at 4 °C and then incubated with secondary antibodies for 1–2 h at RT. The membranes were incubated with ECL solution (Promega, Madison, WI, USA) and imaged using ImageQuant LAS 500 (GE Healthcare, Chicago, IL, USA).
The primary antibodies used were anti-BIS [1 (link)], anti-HSF1, anti-HSP70 (Enzo Life Sciences), anti-GAPDH, anti-HSPB5, anti-YAP1 (Santa Cruz Biotechnology), anti-cleaved PARP1, anti-desmin, anti-HSPB8, anti-p62 (ABCAm), anti-COX4 (Cell Signaling Technology, Danvers, MA, USA), and anti-filamin C (Novus Biologicals, Centennial, CO, USA).

Half a million HeLa cells were seeded in 6-well culture dishes and were treated with different concentrations of ursolic acid (UA), oleanolic acid (OA), or hederagenin (HED) for 5 and 24 h. The medium was then removed and the cells were washed twice with 500 μL of cold PBS (phosphate buffered saline) (4°C) before 300 μL of MPER cell lysis reagent (Thermo Fisher Scientific) was added to each well and incubated for 5 min at 4°C. The wells were scraped to collect the cells, which were transferred to microcentrifuge tubes. Lysed proteins were isolated by centrifugation and quantified using a BCA assay. The same volume of proteins was separated by electrophoresis on a 7.5% polyacrylamide gel (Bio-Rad) and then subjected to electroblotting on a 0.45 µm pore diameter nitrocellulose membrane. The blots were probed with anti-cleaved PARP-1 (poly(ADP-ribose) polymerase-1) (Abcam; 1 : 5000), anti-PARP-1 (Abcam; 1 : 5000), β-actin (Abcam; 1 : 10000), and goat polyclonal antibody to rabbit IgG (HRP) (Abcam; 1 : 5000). Reactive bands were detected by chemiluminescence on a C-DiGit blot scanner (LI-COR Biosciences). Images were captured, stored, and analyzed with the “Image Studio Digits ver. 5.0” program.

HL60 and HL-60R cells were seeded at a density of 1.5 × 10⁵ cells/well in six-well plates for 36 h. Next, the cells were treated with or without 10 μM MK-4, MKH-DMG, or MKH-SUC for 24, 48, or 72 h. The cells were washed with PBS after removing the drug-containing medium and lysed with RIPA buffer (0.5% NP-40, 0.25% sodium deoxycholate, 0.05% SDS, 150 mM NaCl, and 50 mM HEPES, pH 7.4) containing a protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). Each cell lysate was combined with sample loading buffer (Nacalai Tesque), separated on 15% SDS-PAGE gels (SuperSep Ace, FUJIFILM Wako), and transferred onto PVDF membranes (BIO-RAD, Hercules, CA, USA). After blocking, the membranes were incubated with the following primary antibodies: anti-pro/p17-caspase-3, anti-cleaved PARP1 (1:2000) (ab136812; Abcam, Cambridge, UK), rabbit anti-Bak (1:2000) (#12105; Cell Signaling Technology, Danvers, MA, USA), and mouse anti-GAPDH antibody (1:2000) (G8795; Sigma-Aldrich, St. Louis, MO, USA). After washing, the membranes were treated with appropriate secondary antibodies and visualized using Immunostar LD or Immunostar Zeta (FUJIFILM Wako).