Protease inhibitor cocktail set

A total of 2 × 10⁷ BCBL-1 cells were washed with 1× PBS and resuspended in nuclear lysis buffer I (10 mM HEPES pH 7.4, 20 mM KCl, 1.5 mM MgCl₂, 0.5 mM EDTA, 1 mM TCEP, 0.5 mM phenylmethylsulphonyl fluoride [PSMF] and 0.1% NP-40) supplemented with 10 µL of murine RNase Inhibitor (200U final), and 5 µL Protease Inhibitor Cocktail Set (EMD Millipore 539134) in a total volume of 1 mL. Cells were sonicated for 180 sec at 4°C, 15 peak power, 10 duty factor, 200 cycles at 1.5 average power with the wavelength adaptor no. 500534. DNase I (NEB M0303) treatment was performed according to the manufacturer's protocol, followed by 30 min 16,000g centrifugation at 4°C. The whole-cell protein lysates and the proteins cross-linked to PAN were analyzed by 10% SDS-PAGE. Proteins were transferred to a polyvinylidene fluoride (PDVF) membrane using a semidry blotting system (Thermo Fisher IB24002). The membranes were washed with 1× PBS with 0.1% Tween 20 (PBST) and blocked with 4% (w/v) nonfat milk in 1× PBST for 1 h at room temperature. The membranes were incubated with a specific primary antibody (Supplemental Table 2) for 1 h at room temperature, washed thrice with 1× PBST, and incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody (Supplemental Table 2) for 45 min at room temperature. Blots were developed using the enhanced chemiluminescence substrate (Bio-Rad 1705061).

HEK293FT cells were transfected with pCAGGS-HA BRD8 (WT), pCAGGS-HA BRD8 (ΔC), or pCAGGS-HA BRD8 (ΔB) using FuGENE6 (Promega). Forty-eight hours after transfection, the cells were lysed in 0.4% Nonidet P-40 buffer (10 mM Tris-HCl pH7.5, 150 mM NaCl) supplemented with a Protease Inhibitor Cocktail Set (Merck). Pre-cleared cell extracts were incubated with anti-HA (Roche Cat# 11867431001, RRID:AB_390919) or anti-Myc antibody (Sigma-Aldrich Cat# M5546, RRID:AB_260581), followed by the incubation with Protein G-Sepharose beads (Cytiva) at 4°C. The immunocomplexes were then washed with 0.4% Nonidet P-40 buffer and subjected to immunoblotting. To evaluate the interactions between BRD8 and the FOS family members, nuclear fraction (Nuclear Extract Kit, Active Motif, Carlsbad, CA, USA) isolated from HCT116 or SW480 cells was subjected to immunoprecipitation with anti-c-Fos (Cell Signaling Technology, Danvers, MA, USA, Cat# 2250, RRID:AB_2247211), FRA1 (Cell Signaling Technology Cat# 5281, RRID:AB_10557418), or Fra2 (Cell Signaling Technology Cat# 19967, RRID:AB_2722526) antibody, followed by immunoblotting with anti-BRD8 (Sigma-Aldrich Cat# HPA001841, RRID:AB_1845438) or anti-TIP60 antibody (Millipore Cat# DR1041, RRID:AB_2128436).

hTERT-immortalized doxycycline inducible dermal fibroblasts were lysed in IP buffer (20mM Tris-HCl pH7.5, 150mM NaCl, 2mM EGTA, 2mM MgCl2, 0.5% NP-40, 1mM DTT, 1U/ml Benzonase, 1x Protease inhibitor cocktail set from Milipore) for 1h at 4°C and cleared of insoluble material by centrifugation. 1mg of extract was then incubated with 2μg of α-GRP78/BiP rabbit polyclonal antibody (Abcam, ab21685) or 2μg normal rabbit IgG antibody (Cell Signaling, 2729S) overnight, followed by incubation with Pierce protein A/G magnetic beads (Thermo Fisher Scientific) at 4°C for 4h. Beads were thoroughly washed 3x in IP buffer (w/o Benzonase), and precipitated proteins were eluted using 1x Laemmli sample buffer (Bio-Rad), followed by Western blot analysis. Antibody used for Western blot detection was α-GFP rabbit polyclonal (Abcam, ab290) and α-GRP78/BiP rabbit polyclonal (Abcam, ab21685).

Under deep anesthesia, mice were transcardially perfused with ice-cold PBS until the irrigation fluid was completely clear. After decapitation, the brains were removed, and the hippocampus and cortex were micro-dissected on ice in Hank’s balanced salt solution (HBSS) and the meninges were removed. The cell lysis buffer from CST (9803) was diluted with ddH₂O, and 1 mM protease-inhibitor cocktail (set III, EDTA-Free; 539134, Millipore) was added before use; tissues were extracted at a ratio of 100 mg of tissue to 1 mL of buffer, homogenized, and spun for 10 min at 14000 × g at 4°C to remove precipitates. Duplicate samples of the supernatant were collected for quantifying total protein concentrations by using Bradford protein assay. Each sample was analyzed for cytokines by using a MCYTOMAG-70K Mouse Cytokine magnetic kit (EMD Millipore, Billerica, MA, USA), and data were analyzed using Milliplex Analyst 5.1 software (EMD Millipore). Measurements were corrected for noise by subtracting the average of two technical control wells for each secreted soluble factor.