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To analyze localization of exogenous RelA, Ponatinib were co-transfected with plasmids expressing EGFP-RelA and plasmids expressing FLAG-gankyrin or FLAG alone (at a molar ratio of 1:10), and EGFP fluorescence was detected by fluorescence microscopy. More than 100 cells were assessed per slide. For confocal immunofluorescence microscopy, cells were co-transfected with plasmids expressing RelA and FLAG-gankyrin, treated with TNFα for 8 h, and analyzed using mouse anti-RelA antibody (Santa Cruz), rabbit anti-FLAG antibody (Sigma), sheep anti-mouse immunoglobulin antibody conjugated to TRITC (BD PharMingen), and goat anti-rabbit immunoglobulin antibody conjugated to FITC (Dako) as described [8].
Analyses of protein–protein interactions. Lysates were prepared from HeLa and 293 cells expressing HA-tagged gankyrin or HA alone and HeLa cells treated with TNFα. Immunoprecipitation and Western blot analysis were performed as described [9]. For immunoprecipitation, mouse anti-HA (Roche), anti-RelA (Santa Cruz), and anti-p50 (Santa Cruz) antibodies, agarose-conjugated rabbit anti-RelA antibody (Santa Cruz), mouse immunoglobulin (Santa Cruz), and agarose-conjugated rabbit immunoglobulin (Santa Cruz) were used.