Dc detergent compatible protein assay

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.

Cells were plated in 60 mm plates at a density of 1 × 10⁶ cells per plate in regular culture media and cultured for 24 h. Then cells were treated with 1 μM senexin B or solvent control (0.1% DMSO, MilliporeSigma, St. Louis, MO, USA) for 3 h before lysing cells in 0.5 mL RIPA (radio immunoprecipitation assay) lysis buffer with 1× protease inhibitor cocktail. The protein concentration of extracts was determined using the DC (detergent-compatible) protein assay (Bio-Rad Laboratories). Protein (50 μg) was resolved on 4–12% Express-Plus polyacrylamide gels in Tris-MOPS (SDS) running buffer (GenScript, Piscataway, NJ, USA), transferred to the PVDF (polyvinylidene difluoride) membrane, blocked with 5% non-fat milk and incubated with primary antibodies: CDK8 (sc-1521, Santa Cruz Biotechnology, Dallas, TX, USA), CDK19 (HPA007053, MilliporeSigma) and GAPDH (sc-32233, Santa Cruz Biotechnology) followed by either anti-goat (sc-2020, Santa Cruz Biotechnology), anti-rabbit (NA934, GE Healthcare, Chicago, IL, USA) or anti-mouse (NXA931, GE Healthcare) secondary antibodies. Bands were visualized with Western Lighting Plus ECL (enhanced chemiluminescence) detection reagent (Perkin Elmer, Waltham, MA, USA) using ChemiDoc Touch™ (Bio-Rad Laboratories, Hercules, CA, USA). Images were analyzed using ImageLab software (Bio-Rad, Version 5.2.1 build 11).

Cells in culture were collected via trypsinization and pelleted via centrifugation. Cell pellets were lysed in AZ lysis buffer (50mM Tris pH 8, 250mM NaCl, 1% NP-40, 0.1% SDS, 5mM EDTA, 10mM Na4P2O7, 10mM NaF, 1x cOmplete EDTA-free Protease Inhibitor Cocktail (Roche), 1x PhosSTOP (Roche)). The protein concentration of each sample was determined using the DC™ (detergent compatible) protein assay (Bio-Rad Laboratories, Inc.). Protein concentrations were normalized and samples were prepared with 5x Laemmli sample buffer. Samples were run on a gradient gel (Bio-Rad) and transferred for Western blot on 0.45um Nitrocellulose membrane (Bio-Rad).
The primary antibodies used were mouse anti-PTEN (sc7974, Santa Cruz Biotechnology), and mouse anti-Beta Actin (Cell Signaling Technology #4970). Primary antibodies were used at 1:1000 dilutions and were incubated for 1 to 2 hours at room temperature or overnight at 4°C. Secondary goat anti-mouse antibody (Thermo Fisher Scientific/Pierce) or were used at a 1:10,000 dilution for 1 hour at room temperature. Primary and secondary antibodies were prepared in 5% milk. Three washes with Tris Buffered Saline with Tween 20 were each performed after primary incubation and after secondary incubation. Membranes were developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).

Leaf total proteins were extracted as described previously (Hackett et al., 2017). Mature rosette leaves (~50 mg) were excised, frozen in liquid nitrogen, and ground into fine powder with stainless steel beads and TissueLyser II (Qiagen). Freshly made plant protein extraction buffer (50 mM Tris‐HCl, pH 7.5, 150 mM NaCl, 1% Triton X‐100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM DTT, and 1% of plant protease inhibitor cocktail) was added to the frozen powder at 5 μl/mg tissues. The samples were further homogenized with TissueLyser II. The resulting homogenates were centrifuged at >10,000 g for 3 min at 4°C. The supernatants were transferred to new microfuge tubes and centrifuged again at >10,000 g for 3 min at 4°C to remove residual tissue debris. The protein concentrations were determined using the DC (Detergent Compatible) protein assay (Bio‐Rad) with 0 to 1.4 mg/ml of bovine serum albumin as standards.

Cells were lysed using RIPA lysis buffer. Protein concentration was determined using the DC (Detergent compatible) protein assay (BioRad, Hercules, CA, USA). Equal amounts of protein lysates (20 μg) were separated by gel electrophoresis using a NuPAGE Novex 4–12% Bis-Tris Midi Gel (Invitrogen) and transferred to nitrocellulose or polyvinylidene fluoride membranes. Immunoblots were probed using anti-BCL-XL antibody (Cat#2764), anti-BCL2 antibody (Cat#4223) from Cell Signaling Technology (Danvers, MA), or anti-β-actin (sc-47778) from Cell Santa Cruz Biotechnology (Dallas, TX).