Bolt 4 to 12 bis tris plus gel

All procedures for sample preparation were performed at 4 °C. Skin was taken from adult mice (Table S1) and proteins were extracted using RIPA buffer (Thermo Fisher Scientific) containing Halt Protease Inhibitor Cocktail, EDTA-Free (Thermo Fisher Scientific). After centrifugation, soluble proteins in the extract were mixed with SDS sample buffer with reducing agents. These protein solutions were separated on Bolt 4 to 12% Bis-Tris Plus Gels (Thermo Fisher Scientific), then transferred to PVDF membranes, and western blots were performed using antibodies and gel running buffers listed in Table S3. Blots were developed with HRP enhanced SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and detected by ChemiDoc MP imaging system (BioRad) using the software Image Lab version 4.0.1 (BioRad). The intensities of protein signals were measured by Image J.

Cells were lysed in 1× Laemmli buffer (62.5 mM Tris-HCl [pH 6.8], 10% glycerol, 2% SDS, 100 mM dithiothreitol [DTT], and 0.02% bromophenol blue), and cell lysates were homogenized with QIAshredder columns (Qiagen). Samples were boiled at 95°C for 5 min before they were loaded on Bolt 4 to 12% Bis-Tris plus gels (Thermo Fisher Scientific) to perform protein separation by gel electrophoresis. After 20 to 35 min at 200 V, proteins were transferred to nitrocellulose membranes (GE Healthcare Life Sciences) for 70 min at 10 V. Next, membranes were blocked in 5% milk in Tris-buffered saline (20 mM Tris-HCl [pH 7.6] and 150 mM NaCl) with 0.1% Tween 20 (Sigma-Aldrich) (TBS-T) for 1 h at room temperature (RT) before membranes were incubated with appropriate dilutions of primary antibodies in TBS-T either for 2 h at RT or overnight at 4°C. After incubation with conjugated secondary antibodies in TBS-T, signals were developed using an LAS-4000 mini (Fujifilm Life Sciences) or an Odyssey Fc imaging system (LI-COR Biosciences). Signal intensities were quantified using the Image Studio Light software (LI-COR Biosciences).

Cells were harvested and lysed in lithium dodecyl sulfate (LDS) sample buffer (Invitrogen) at 5 million cells/ml, separated electrophoretically using Bolt 4 to 12% bis-tris plus gels (Invitrogen), and transferred onto polyvinylidene difluoride (PVDF) membranes (0.2 μm in pore size) using PVDF Mini Stacks and iBlot 2 (Invitrogen). Membranes were immersed in 5% bovine serum albumin (Thermo Fisher Scientific) and then probed with primary antibodies against SGF29 (1:1000; HPA052590, Sigma), H3K9ac (1:1000; 61663, Active Motif), H3K27ac (1:1000; 91193, Active Motif), KAT2A (1:1000; 3305S, Cell Signaling Technology Inc.), KAT2B (1:1000; 3378S, Cell Signaling Technology Inc.), RPL8 (1:1000; HPA050165, Sigma), RPS2 (1:1000; PIPA530160, Thermo Fisher Scientific), and Histone H3 (1:1000; 4499, Cell Signaling Technology) at 4°C overnight. After washing, the membranes were incubated with horseradish peroxidase–conjugated goat anti-rabbit (1:200,000; 31460, Invitrogen), anti-mouse (1:200,000; 31430, Invitrogen), and anti-rat (1:200,000; 31470, Invitrogen) immunoglobulin G antibodies at room temperature for 1 hour. Chemiluminescent signals were developed using the SuperSignal West Femto Substrate (Thermo Fisher Scientific) and detected using a ChemiDoc imaging system (Bio-Rad).

Cells were harvested and lysed in lithium dodecyl sulfate sample buffer (Invitrogen) at 5 × 10⁶ cells/ml, separated electrophoretically using Bolt 4 to 12% bis-tris plus gels (Invitrogen), and transferred onto polyvinylidene difluoride (PVDF) membranes (0.2 μm pore size) using PVDF Mini Stacks and iBlot 2 (Invitrogen). Membranes were immersed in 5% nonfat milk then probed with rabbit antibodies against ACTR5 (sc-376364, Santa Cruz Biotechnology; 1:1000), IES6 (PA5-61869, Thermo Fisher Scientific; 1:1000), CDKN2A (ab108349, Abcam; 1:1000), CDK6 (ab124821, Abcam; 1:1000), E2F1 (3742S, Cell Signaling Technology; 1:1000), Rb (ab181616, Abcam; 1:1000), phospho-S780 Rb (ab173289, Abcam; 1:1000), and β-actin (4970S, Cell Signaling Technology; 1:1000) at 4°C overnight. After washing, the membranes were incubated with horseradish peroxidase–linked goat anti-rabbit immunoglobulin G antibody (31460, Invitrogen; 1: 200,000) at room temperature for 1 hour. Chemiluminescent signals were developed using the SuperSignal West Femto Substrate (Thermo Fisher Scientific) and detected using a ChemiDoc imaging system (Bio-Rad).

Protein samples were separated on a Bolt 4 to 12% Bis-Tris Plus gel (Invitrogen), blotted onto a polyvinylidene difluoride (PVDF) membrane (Amersham), blocked in 5% milk, and probed with the appropriate primary antibody listed in Table S2 diluted in 5% milk in phosphate-buffered saline plus Tween (PBS-T). This was followed by incubation with a secondary horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse antibody (1:2,000; Bio-Rad), depending on the origin of the primary antibody. Proteins were visualized using the Pierce ECL system (Genetica/Bio-Rad) on a ChemiDoc instrument (Bio-Rad).