Ab10485

Cells were lysed in LDS sample buffer and 50 µg total protein was separated either on 3–8% Tris-Acetate gels (Thermo Fisher Scientific) for analysis of p300 and CBP expression or 4–12% Bis-Tris gels (Expedeon, San Diego, CA, USA) for all other proteins and transferred onto methanol-activated PVDF membranes or 0.2 µm nitrocellulose membranes (GE Healthcare). Blocking of membranes and antibody incubation were performed in 5% BSA in TBS-T. The following antibodies were used: anti-p300 (1:4000, ab10485, Abcam), anti-CBP (1:1000, Cell Signaling Technology), anti-c-Myc (1:1000, Cell Signaling Technology), anti-Histone H3 (1:1000, Cell Signaling Technology), anti-Acetyl-Histone H3 (Lys18, 1:1000, Cell Signaling Technology), anti-Vimentin (1:500, Santa Cruz Biotechnology), anti-Vinculin (1:500, Santa Cruz Biotechnology), anti-Lamin A (1:2000, Abcam), anti-α-tubulin (1:500, Santa Cruz Biotechnology), and anti-GAPDH (1:50000, Merck Millipore). House-keeping controls were selected in a cell line-specific manner on the basis of data showing no change in their expression in that cell line.

Paraffin-embedded tissue samples were collected, cut into 3-μm thick sections and fixed on silicified slides. After deparaffined and rehydrated, the sections were heated in 0.01 mol/L saline citrate buffer at 96°C to 98°C for 15 minutes to unmask antigens, treated with 3% hydrogen peroxide for 15 minutes at room temperature to inactive endogenous peroxidase and incubated with 10% goat serum for 30 minutes at room temperature to block nonspecific binding. Then slides were incubated overnight at 4°C with a rabbit monoclonal to β-arrestin1 antibody (Abcam Biotechnology, ab32099; diluted, 1:200) or a rabbit polyclonal to p300 antibody (Abcam Biotechnology, ab10485; diluted, 1:1000). The subsequent steps were according to the instructions of Zymed (streptavidin-peroxidase method). The sections were then counterstained with hematoxylin before dehydration and mounting. For negative control, we carried out the same steps as described previously while the antibody was replaced by phosphate-buffered saline.

The binding between DLX6‐AS1 and p300 protein was detected by the RIP kit (Millipore, Temecula, CA, USA). Cells were washed with pre‐cold PBS, and the supernatant was discarded. RIPA lysis (P0013B, Beyotime Biotechnology Co., Ltd., Shanghai, China) of equal volume was added to lyse cells in the ice‐bath for 5 minutes. The supernatant was obtained through centrifugation (7043.4 g, 4ºC, 10 minutes). A portion of the cell extract was used as Input and the other part was used for coprecipitation with antibody. Magnetic beads (50 μL) were washed, re‐suspended in 100 μL RIP Wash Buffer and incubated with the corresponding antibodies (5 μg), including rabbit antibody to p300 (1:100, ab10485, Abcam Inc, Cambridge, MA, USA) for a 30‐minutes incubation at room temperature, where rabbit anti‐human antibody to immunoglobulin G (IgG) (1:100, ab109489, Abcam Inc, Cambridge, MA, USA) served as NC. The magnetic bead‐antibody complex was washed, re‐suspended in 900 μL RIP Wash Buffer and incubated with 100 μL cell extract at 4ºC overnight. The sample was washed three times and placed on the magnetic base for collection of the magnetic bead‐protein complex. Following detachment by protease K, RNA was extracted from each sample and its Input for further DLX6‐AS1 quantification by RT‐qPCR.¹⁴