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. 2015 May 14;10(5):e0127334. doi: 10.1371/journal.pone.0127334

Fig 2. Overexpression of GITR impacts TNFα-induced non-canonical NF-κB activation.

Fig 2

A. MM1.S, OPM1 (GITR low) and RPMI8226 (GITR high) cells were exposed to TNFα (10ng/ml) for 60 minutes. NF-κB activity was evaluated by DNA binding ELISA assay. NF-κB p65 transcription factor binding to its consensus sequence on the plate-bound oligo nucleotide was examined from nuclear extracts. Data represent mean ± SD of triplicate experiments. *P<0.05, **P<0.01 and ***P<0.001 compared with indicated groups. B. MM1.S, OPM1 and RPMI8226 cells were exposed to TNFα (10ng/ml) for 60 minutes. Nuclear and whole cell lysates were subjected to western blot using anti-p65, -p52 and Actin antibodies. C. Cells were exposed to TNFα (10ng/ml) for 15, 30, or 60 minutes. Nuclear protein and cytoplasmic extraction were subjected to western blot using anti-p52, -RelB and -nucleolin antibodies. D. pCMV-GITR and GITR-MM1.S cells were harvested at 24 hours after treatment with and without TNF-α (10ng/ml) for 60 minutes. Immunocytochemical analysis was assessed using anti-phospho-NF-κB-p52 antibody, with DAPI used to stain nuclei.