For localization of endogenous p65 in MEFs, WT and GSK-3β-null MEFs were
grown on coverslips for 24 h and stimulated with 2 μg/ml
of TNFα for the indicated periods of time. Cells were fixed,
permeabilized, stained with rabbit monoclonal anti-p65 (1 : 300 dilution)
and analyzed via confocal microscopy as previously described.53 (link) To detect subcellular distribution of GSK-3β,
Panc04.03 cells were transfected with in pCMS4-H1P-eGFP- GSK-3β vectors by
electroporation and grown on coverslips for 48 h before staining with mouse
monoclonal anti-flag M2 antibody (1 : 800). For PLA detection of
p50/p65 interaction in situ, we used the Duolink in situ PLA kit
from Olink Bioscience according to the supplier's instructions. Mouse mAb against
p50 (SC8414, Santa Cruz Biotechnology) and rabbit mAb against p65 (#8242, Cell
Signaling Technology) were used as primary antibodies at dilutions of
1 : 50 and 1 : 300, respectively. The anti-rabbit plus and
anti-mouse minus secondary antibodies were used as PLA probes and Texas red as detection
regent (Supplementary information).