Dmi6000b series fluorescent microscope

Immunofluorescence assays were carried out as previously described¹¹. 1 × 10⁵ HEK293 cells overexpressing Flag-WT-YBX1 were seeded in a 24-well plate unto a 0.1% sterile gelatin pre-coated coverslip/well and left overnight. Cells were fixed with 4% formaldehyde solution for 30 min followed by blocking buffer for 10 min at R.T. Coverslips were further probed with anti-Flag antibody for the detection of Flag-tagged WT-YBX1 and anti-PRMT5 antibody for the detection of endogenous PRMT5 followed by Alexa Fluor 488 (green) goat anti-mouse IgG and Alexa Fluor 594 goat anti-rabbit IgG. Coverslips were sealed using mounting media containing HOECHST to stain the nucleus. All slides were visualized under a Leica DMI6000B series fluorescent microscope at 63 × magnification.

1 × 10⁵ PANC1 or MIA PaCa2 cells were seeded onto coverslips in a 24-well plate. The next day, the cells were treated with or without IL-1β for 1 h. After treatment, the cells were fixed with 4% formaldehyde for 30 min, and the reaction was quenched by 100 mM glycine for 5 min. The cells were gently washed with 1× PBS and then blocked and permeabilized with blocking buffer (1XPBST and 1% BSA) and permeabilizing buffer (Blocking buffer with 0.2% Tritonx-100), respectively. Cells were further probed with anti-FLAG antibodies for FLAG-tagged WT-p65 or S550A, S551A, and S550A/S551A and Alexa Fluor 488 (green) goat antimouse IgG. Before sealing the coverslips, mounting medium with DAPI was used to stain the nucleus. The slides were examined under a Leica DMI6000B series fluorescent microscope at 40× magnification.