Effect of GITR on canonical NF-κB pathway.

A) Effect of GITR on TNF-α induced NF-κB activation. NF-κB activity has been evaluated by using DNA binding ELISA assay in GITR- and GITR+ MM1.S cells. Cells were exposed to TNF-α (10ng/mL) for 15-30-60 minutes. NF-κB p65 and p50 transcription factor binding to its consensus sequence on the plate-bound oligo nucleotide was examined from nuclear extracts. Data represent mean plus or minus SD of triplicate experiments. Nuclear protein lysates were also subjected to Western blot using anti-p50, -p65 and -nucleolin antibodies. B) GITR- and GITR+ were harvested at 24 hours after treatment with and without TNF-α (10 ng/mL) for 60 minutes; Immunocytochemical analysis was assessed using anti-phospho-NF-κB-p50 antibody, with DAPI used to stain nuclei. C) GITR- and GITR+ cells were exposed to TNF-α (10ng/mL) for 15-30-60 minutes. Whole cellular protein lysates have been subjected to Western blot using anti-p65, -phospho(p)-IκB beta, -IκB beta, -IκB alpha, and -actin antibodies. D) GITR- and GITR+ cells were exposed to TNF-α (2.5-5-10ng/mL) for 16 hours. Whole cellular protein lysates have been subjected to Western blot using anti-BCL-2, -survivin and -actin antibodies. E) GITR- and GITR+ cells were exposed to TNF-α (2.5-10ng/mL) for 12 hours. Apoptosis was evaluated by Annexin/PI staining and flow cytometry analysis. Mean±SD.