Western blot analysis of BRD4. 50 µg total protein lysate from human embryonic kidney HEK293T cells (lane 2) and mouse myoblast C2C12 cells (lane 3) in 1X NuPAGE® LDS Sample Buffer (Product # NP0007) + 1mM DTT loaded onto a NuPAGE™ Novex™ 4-12% Bis-Tris protein gel (Product # NP0321PK2). Molecular weight marker loaded for size reference (lane 1). Gel run in 1X NuPAGE® MOPS SDS Running Buffer (Product # NP0001) at room temperature and then transferred to a methanol-activated Novex™ PVDF membrane (Product # LC2002) using a Invitrogen Mini Blot Module (Product # B1000) in 1X NuPAGE® Transfer Buffer (Product # NP0006) at 20V for 2 hours at room temperature. Membrane was rinsed in 1X TBS-T and incubated in blocking solution (5% milk in 1X TBS-T) for 1 hour at room temperature on a rocking platform. HDAC1 detection was performed by incubating blot with rabbit polyclonal anti-BRD4 antibody (Product # PA5-41550; 0.9 mg/mL) diluted at 1:1000 in blocking solution for 1 hour at room temperature on a rocking platform. Primary antibody was washed 10 times with 1X TBS-T over the course of 1 hour at room temperature on a rocking platform. Secondary antibody binding was performed by incubation with goat anti-rabbit IgG-HRP secondary diluted at 1:5000 in blocking solution for 1 hour at room temperature on a rocking platform. Following washes (as above), chemiluminescent imaging was performed. Data courtesy of Dr. Raj Jain's laboratory.