Ab50702

Embryos were lysed with 1% Triton-X100 in phosphate-buffered saline containing a protease inhibitor cocktail. For immunoprecipitation analysis, 600–1200 μg of protein was incubated with the anti-CBP antibody (Abcam ab50702, UK) or IgG and protein A/G plus agarose beads (Santa Cruz sc-2003, USA) overnight at 4°C. The immunoprecipitation reactions were washed five times and boiled for western blotting. Approximately 80–120 μg of protein was resolved on 10% SDS-PAGE gels and transferred to 0.45 μm PVDF membranes. All membranes were blocked in 5% marvel milk in TBST and then incubated with appropriate antibodies at 4°C overnight. Primary antibodies were used to detect RARα (Novus NBP2-45516, USA) and CBP (Novus NB100-91721).

The RNA immunoprecipitation assay was performed basically using the protocol of Peritz et al.^{52 (link)} with some modifications. Briefly, HSV-1 or Mock infected HeLa cells were fixed with 1% formaldehyde and sonicated on ice for 2 cycles at 50% power for 15 s with 2 min pauses between each cycle using a probe sonicator (Sonics & Materials, Inc.). Then, cell lysates were incubated with anti-SRSF2 antibodies (Abcam, ab11826), anti-P300 antibodies (Abcam, ab59240), anti-CBP antibodies (Abcam, ab50702), anti-EZH2 antibodies (Abcam, ab186006), or anti-IgG antibodies (ab205718 or ab205719) at 4 °C overnight. RNA enriched from the immunoprecipitation was retrotranscribed for the real-time PCR. The primers for RT-PCR analysis are listed in Supplementary Table 1.

Western blot were carried out as previously described [18 (link)]. Primary antibodies against E-cadherin (ab40772), N-cadherin (ab76057), Vimentin (ab8978), β-catenin (ab16051), CBP (ab50702), BRG1 (ab108318), MYC (ab9106) and GAPDH (ab245356) and secondary antibodies were all obtained from Abcam.

To investigate the interaction between Aβ and CAV2, TGFB2 or TGFBR1, U251 cells were inoculated with β-amyloid (1–42), HiLyte Fluor™ 488-labelled (ANASPEC, AS-60479-01). After 1 h, the cells were washed, fixed and incubated with CAV2 (Abcam, ab133484), TGFB2 (Abcam, ab36495) or TGFBR1 antibodies (Abcam, ab31013). The cells were washed, counterstained with DAPI and observed under an Olympus FV1000 confocal laser microscope. To study the interaction between NEAT1 and P300/CBP, U251 cells were incubated with the NEAT1 probe overnight at 37 °C and then the anti-P300 (Abcam, ab59240) or anti-CBP antibodies (Abcam, ab50702) for 1.5 h at room temperature. After the cells were washed and incubated with the secondary antibody, they were counterstained with DAPI and mounted for observation. Cell images were obtained with an Olympus FV1000 confocal microscope. To study the role of NEAT1 in the interaction between STAT3 and H3K27Ac, U251 cells were transfected with a NEAT1 or negative control siRNA for 36 h and then incubated with the anti-STAT3 Y705 (Abcam, ab76315) and anti-H3K27Ac antibodies (Abcam, ab4729) for 1.5 h at room temperature. After the cells were washed and incubated with the secondary antibody, they were counterstained with DAPI and mounted for observation. Cell images were obtained with an Olympus FV1000 confocal microscope.