Cytobuster reagent

Total proteins were extracted with CytoBuster reagent (Merck) containing protease and phosphatase inhibitor (Roche). Nuclear and cytoplasmic proteins were extracted with Nuclear and Cytoplasmic Extraction reagent (ThermoFisher). For western blot, proteins (10–30 µg) were separated on 10% SDS-PAGE and transferred onto a PVDF membrane. After blocking in 5% BSA in TBS-T (150 mmol/L Tris-HCl, pH 7.4, 50 mmol/L NaCl, 0.1% Tween 20), membranes were incubated with primary antibodies overnight at 4 °C, followed by secondary antibodies for 1 h at room temperature. Bands were visualized with Clarity Western ECL (Bio-Rad) in ChemiDoc XRS+ (Bio-Rad) using Image Lab software v6.1.0 (Bio-rad). The antibody list was shown in Table S5.

Protein was isolated using Cytobuster reagent (Merck, Kenilworth, NJ, USA, 71009-3) supplemented with protease and phosphatase inhibitors (Roche, Basel, Switzerland, 05892791001 and 04906837001, respectively). Cells were mechanically lysed, and centrifuged (13,000 rpm, 4 °C, 10 min). Supernatants were used to quantify protein concentration via the bicinchoninic acid assay (BCA). Proteins were standardized to 1 mg/mL. The expression of KDM5B (Abcam, Cambridge, UK, ab19884), H3K4me3 (Abcam, Cambridge, UK, ab12209), PTEN (Cell Signalling Technologies, Danvers, MA, USA, 9552S), p-ser473-AKT (Cell Signaling Technologies, Danvers, MA, USA, 9271S), AKT (Cell Signaling Technologies, Danvers, MA, USA 9272S), PI3K (Cell Signaling Technologies, Danvers, MA, USA, 4249S), p-ser280-CHK1 (Cell Signaling Technologies, Danvers, MA, USA, 23475), and CHK1 (Cell Signaling Technologies, Danvers, MA, USA, 2360S) were determined using Western blotting as previously described [43 (link)]. The Image Lab Software version 5.0 (Bio-Rad, Hercules, CA, USA) was used to measure band densities of expressed proteins. Protein expression is represented as relative band density and calculated by normalizing the protein of interest against the housekeeping protein, β-actin.