Owl hep 1

PC3 cells were cultivated with an IC₅₀ dose of 8 for 2 h, 6 h, 12 h, 24 h, and 48 h. The cell lysis was performed using protein lysis buffer (62.5 mMTris–HCl (pH 6.8), 2% (w/v) SDS, 10% glycerol, and 50 mM dithiothreitol). The proteins were electrically separated using 12% SDS-polyacrylamide gels where a PageRuler prestained ladder was used as a protein molecular weight marker. The proteins were electrically transferred to nitrocellulose membranes by western blot system (Owl HEP-1, ThermoFisher Scientific, Schwerte, Germany). The membranes were blocked by 5% (w/v) BSA in PBS with 0.1% Tween 20 for 1 h at RT. Afterwards, blots were incubated overnight at 4 °C with α/β-Tubulin rabbit Ab, Caspase-3 rabbit Ab, β-actin rabbit Ab, and Bcl-XL rabbit Ab. As a secondary antibody Anti-rabbit IgG, HRP-linked Antibody was used. Bands were visualized using an ECL Prime Western Blotting System.

PC3 cells were cultivated with an IC₅₀ dose of compound 4 for 2 h, 6 h, 12 h, 24 h and 48 h. Protein lysis buffer (62.5 mM Tris–HCl (pH 6.8), 2% (w/v) SDS, 10% glycerol, and 50 mM dithiothreitol) was used to lysis the cells. The electrical separation of the isolated proteins was performed using 12% SDS-polyacrylamide gels where a PageRuler prestained ladder was used as protein molecular weight marker. The proteins were electrically transferred to nitrocellulose membranes by a western blot system (Owl HEP-1, Thermofisher Scientific, Waltham, MA, USA). The membranes were blocked with 5% (w/v) BSA in PBS with 0.1% Tween 20 for 1 h at RT. Afterwards, Blots were incubated over night at 4 °C with p38 MAPK rabbit Ab, α/β- Tubulin rabbit Ab, p70 S6 Kinase rabbit Ab, Caspase-3 rabbit Ab, P-p38 MAPK (T100/Y182) rabbit Ab, P-p70 S6 Kinase (T389) rabbit Ab or PARP rabbit Ab, LC3A/B (D3U4C) XP^® Rabbit mAb. As a secondary antibody Anti-rabbit IgG, HRP- linked antibody was used. Bands were visualized using an ECL Prime Western Blotting System.