Fig 2: TRAIL-R1 and TRAIL-R2 are enriched in the chromatin fraction upon TRAIL stimulation. (A) PancTu-I cells were stimulated with 100 ng TRAIL for one hour. The obtained chromatin fractions (Chr) were analyzed by Western blotting and compared to the supernatant (SN) and to the whole cell lysate (WL). Markers for different subcellular fraction have been included as follows, ß-Tubulin (cytoplasmic marker), RNA polymerase 2A (POLR2A) and Histone H3 (chromatin markers), Lamin A/C (nuclear marker). Band intensities of nTRAIL-R1 and nTRAIL-R2 bands were analyzed by densitometry in relation to POL2A and normalized to untreated controls. (B) Co-localization of TRAIL-R1 or TRAIL-R2 with Histone H3 in PancTu-I cells was studied by immunofluorescence and confocal LSM. (C,D) Plasma membrane proteins of PancTu-I and Panc89 cells were labeled with biotin for 1 h at 4 °C, followed by 1 h incubation at 37 °C with or without stimulation with TRAIL in indicated concentrations. Chromatin isolation was done as described in Material and Methods. Biotinylated proteins purified from chromatin fractions were analyzed in Western blot for the presence of TRAIL-R1 and TRAIL-R2. Lysates from chromatin fractions were immunoblotted for nuclear markers (POLR2A and Histone H3) as control for the equal amounts of extracts used for purification of biotinylated-proteins.