Fig. 3

Verification of hnRNPD binding partners in oral cancer. A, i 14-3-3 binding motif of hnRNPD. Photomicrograph showing presence of 14-3-3 binding motif, Mode 1 in hnRNPD polypeptide sequence as revealed by http://www.motif.scan.mit.edu; ii Western blot showing presence of 14-3-3ζ and hnRNPD proteins in immunoprecipitates of hnRNPD/14-3-3ζ obtained from OSCC cells (SCC4 and MDA1986) while no band was seen in negative controls (i.e. beads only control). Input represents whole cell lysates from MDA1986 used as positive control for Western blotting; iii Confocal laser scan microscopy (CLSM) demonstrating nuclear and cytoplasmic expression of 14-3-3ζ (green, FITC) and hnRNPD (red, TRITC) in oral cancer cells a SCC4 and b MDA1986 cells. Blue colored DAPI was used as nuclear stain. Merged photomicrographs represent the co-localization of hnRNPD with 14-3-3ζ in oral cancer cells; iv Confirmation of binding of 14-3-3ζ at p-Ser83 of hnRNPD in oral cancer cells. Western blot revealed the presence of p-Ser83 of hnRNPD in immunoprecipitates obtained by using anti-14-3-3ζ antibody from SCC4 and MDA1986 cell line; B Western blot showing presence of hnRNPK and S100A9 proteins in immunoprecipitates of hnRNPD obtained from OSCC cells (SCC4 and MDA1986) while no band was seen in negative controls. Input represents whole cell lysates from MDA1986 used as positive controls; ii Confocal laser scan microscopy (CLSM) demonstrating nuclear and cytoplasmic expression of hnRNPK (green, FITC) and hnRNPD (red, TRITC) in oral cancer cells a SCC4 and b MDA1986 cells. Blue colored DAPI was used as nuclear stain. Merged photomicrographs represent the co-localization of hnRNPD with hnRNPK in oral cancer cells