Immobilon western chemiluminescent hrp

Twenty-four to 48 h posttransfection, cells were washed once with PBS and lysed with PBS buffer with 0.2% NP-40 and protease inhibitor cocktail (Roche). Total protein was measured with a bicinchoninic acid (BCA) protein assay kit. For immunoprecipitation assays, supernatants were incubated with immunoprecipitating antibody overnight at 4°C. Protein A- agarose beads were then used to capture the complexes for 2 h at room temperature or overnight at 4°C, followed by three washes with PBS–0.1% NP-40. Immunoprecipitants were eluted with SDS sample buffer at 100°C for 5 min. Protein samples were separated on a 12.5% SDS-PAGE gel and transferred to nitrocellulose. The membrane was blocked for 1 h at room temperature or overnight at 4°C in 5% milk or 4% BSA in PBS–0.05% Tween 20, followed by incubation with primary antibodies overnight at 4°C. The membrane was washed with PBS–0.05% Tween and probed with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. The membranes were stained with Immobilon Western chemiluminescent HRP (Millipore) or Femto SuperSignal, and quantitation was processed with ImageJ software.

The pool of BRD4 siRNA (smart pool) was obtained from Santacruz with catalog number sc-43,639. The HIV_GKO plasmid was a gift from Eric Verdin (Addgene plasmid # 112,234). HIV_GKO and VSVG plasmid was used to transfect 293T cells using lipofectamine 3000 transfection reagent (catalog number: L3000001) according to the manufacturer’s protocol. For siRNA-mediated knockdown of BRD4, differentiated THP-1 was transfected by nucleoporation using Lonza nucleofector according to the manufacturer’s protocol. Forty-eight hours post-transfection, cells were washed twice with 1X PBS and lysed with 1X PBS buffer with 0.2% NP40 and protease inhibitor cocktail (Roche). Total protein was measured with a BCA protein assay kit. Protein samples were separated on a 10% SDS-PAGE and transferred to nitrocellulose. The membrane was blocked for 1 h at room temperature in 4% BSA in PBS, 0.05% Tween 20 followed by incubation with primary antibodies overnight at 4 °C. The membrane was washed with 1X PBS, 0.05% Tween 20, and probed with HRP-conjugated secondary antibodies at room temperature for 1 h. The membranes were then stained with Immobilon Western Chemiluminescent HRP (Millipore).

Cells were collected and lysed in RIPA buffer, and analyzed by western blot as we described (13 (link), 75 (link)). Briefly, protein concentration was determined with Pierce BCA assay, and Western blot was conducted using Immobilon Western Chemiluminescent HRP (EMD Millipore) and quantified using the Thermo Fisher iBright Imaging system. Nuclear and cytoplasmic fractionation was performed using HAEC lysates with NE-PER^® Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific 78833) per the manufacturer’s instruction. Antibodies used are listed in Table 3.

Cell pellets were lysed in RIPA lysis buffer (50 mM Tris buffer, pH 7.4, 100 mM NaF; 120 mM NaCl, 0.5% NP-40, 100 μM Na₃VO₄) with 1X protease inhibitor cocktail (Fermentas, R1329). Patient tumor and adjoining normal tissue were also homogenised in RIPA lysis buffer with 1X protease inhibitor cocktail. The protein was collected by centrifugation at 13,000 rpm for 10 min at 4 °C, and quantified using bicinchoninic acid (BCA) protein estimation kit (ThermoScientific, 23227). Using SDS-PAGE, ~20 μg of cell lysates or ~25 μg of patient tissue samples were resolved on 10 or 15% gel for separation, and transferred to the PVDF membrane (Merck, IPVH00010). Immunostaining was done using CERS1 (1:5000 dilution) (Abnova, H00010715-A01), CERS2 (1:400 dilution) (SantaCruz, sc-390745), CERS5 (1:2000 dilution) (Abcam, ab73289), CERS6 (1:5000 dilution) (Abnova, H00253782-M01), or β-actin (1:10,000 dilution) (Sigma, A5441) by overnight incubation at 4 °C in 5% BSA in TBST solution (1X TBS; 0.1% Tween 20). After washing, the blots were incubated with goat anti-mouse IgG (L + H) secondary antibody (1:10,000 dilution) (Abcam, ab6789) or anti-rabbit IgG HRP secondary antibody (1:10,000 dilution) (SantaCruz, sc-2004) for 1 h. Exposed X-ray sheets (Carestream, 6568307) were developed using Immobilon Western Chemiluminescent HRP (Merck Millipore, WBKLS0500).