Ab133484

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.

The cells were lysed in ice-cold whole cell extract buffer B (50 mM TRIS–HCl, pH 8.0, 4 M urea and 1% Triton X-100) supplemented with a complete protease inhibitor mixture. The cell extracts were resolved by SDS-PAGE and analysed by western blotting. Protein bands were visualized using ECL Blotting Detection Reagents. The antibodies used for western blotting include CAV2 (Abcam, ab133484), TGFB2 (Abcam, ab36495), TGFBR1 (Abcam, ab31013) and β-actin antibodies (Proteintech, 60008-1-Ig).