Ab27780

The BCL3‐induced enrichment of LINC00176 was determined using a RIP Kit (Millipore). After a pre‐cooled PBS rinse, the HO8910 cells were lysed with equal volume of lysis in ice bath for 5 minutes. The supernatant was attained through centrifugation at 21 912 × g and 4°C for 10 minutes. A portion of the cell extract was used as input, while the remaining was probed with the BCL3 antibody (ab27780, Abcam lnc) for coprecipitation. Immunoglobulin G (IgG) antibody (ab2410, Abcam Inc) served as NC. RNA was extracted from the sample and input after protease K detachment, followed by RT‐qPCR detection.

HO8910 cells were fixed using formaldehyde solution for 10 minutes to initiate DNA‐protein cross‐link. Chromatin fragments were obtained by application of ultrasonic sound (10 seconds each time at an interval of 10 seconds, 15 times in total). The supernatant was collected through centrifugation at 12 000 × g and 4°C for 10 minutes, subpacked into 2 tubes, and incubated with 2 µg NC antibody to IgG (ab2410, Abcam Inc) and 2 µg target protein‐specific antibody to BCL3 (ab27780, Abcam Inc) and p50 (sc‐166588, Santa Cruz Biotechnology, Inc) at 4°C overnight. The DNA‐protein complex was precipitated using protein agarose/Sepharose, followed by centrifugation at 16 099 × g for 5 minutes. The supernatant was discarded, and the non‐specific complex was washed. De‐cross‐linking was conducted at 65°C overnight. DNA fragment was purified and extracted using phenol/chloroform. The binding of BCL3 to the CP promoter was determined by RT‐qPCR using CP‐specific primer.

Cells in each group were lysed with lysis buffer containing 50 mmol/L Tris‐HCl (pH = 7.4), 150 mmol/L NaCl, 10% glycerol, 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 0.5% NP‐40 and protease inhibitor mixture, and then centrifuged, followed by removal of cell fragments. Next, the removed cell lysate was incubated with 1 µg rabbit polyclonal antibody to BCL3 (ab27780, Abcam Inc) and 15 µg protein A/G beads (Santa Cruz Biotechnology, Inc) for 2 hours. After extensive washing, the beads were boiled for 5 minutes at 100°C. The protein was separated using sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and transferred onto the nitrocellulose membrane (Merck Millipore), followed by application of Western blot analysis.