Regulation of PERK Signaling and Leukemic Cell Survival by a Novel Cytosolic Isoform of the UPR Regulator GRP78/BiP
Figure 3
Characterization of human GRP78va protein.
A. Schematic diagram comparing the protein domains of canonical GRP78 and GRP78va. The carboxy-terminal region recognized by the anti-GRP78 monoclonal antibody (Ab) is indicated. B. Schematic diagram of the indicated GRP78va expression plasmids. The base mutations in the 5′ splice site of intron 1 in pcDNA/Grp78va-sm are indicated in red. C. Coupled in vitro transcription and translation assay to detect the protein products from pcDNA/Grp78va and pcDNA/Grp78va-sm plasmids. D. Detection of endogenous GRP78va protein in human cell lines. The post-nuclear (PN) fractions from non-treated or Tg-treated HeLa, HCT116 and HL-60 cells were subjected to Western blot with the anti-GRP78 monoclonal antibody and β-actin served as loading control. GRP78va expression in 293T cells transfected with pcDNA/Grp78va-sm was used as positive control. A darker exposure of the GRP78va bands is shown below the entire blot. E. Immunofluorescence staining of GRP78 and GRP78va. HeLa cells were transfected with pcDNA-Grp78 (left panel) or pcDNA/HA-Grp78va (right panel). The cells were stained with anti-GRP78 antibody (H-129) or anti-HA antibody as indicated, followed by FITC-conjugated secondary antibodies. The confocal immunofluorescence images are shown. F. Cytosolic localization of GRP78va determined by the cell permeabilization assay. HeLa cells transiently transfected with pcDNA/HA-GRP78va were treated with 0.01% digitonin for 5 minutes. Intact cells, digitonin-permeabilized cells, and the released fraction were analyzed by Western blots for the proteins as indicated.