Exploring the Functional Complementation between Grp94 and Hsp90
Fig 6
GRP94 client expression with Grp94/Hsp90 chimeras.
A) Schematic representation of constructs tested. All constructs contained the Grp94 signal sequence (ss), the preN-domain, and the KDEL ER retention sequence found in the Grp94 C-terminal extension (Cx). The FLAG tag is shown as a red bar. B) Detail of the FLAG tag incorporated into each tested construct. C) EGFP gating and intracellular staining of Grp94-null E4.126 cells virally transduced with Grp94 constructs. Top panel: dot plot of the side scattering intensity (SSC-A, Y-axis) of analyzed cells (a measure of cell size and clustering) vs. the fluorescence of the co-expressed EGFP (X-axis). The number of events is represented by the color of the dots. The dots in the magenta box correspond to EGFP positive cells and the numbers show the percentage of all cells that were assigned to this gate. Lower panel: Histogram of the normalized cell count vs. conjugated anti-FLAG (blue) or isotype control (red) fluorescence. Fluorescence intensity is proportional to the copy number of the antibody target. Numbers represent the mean fluorescence intensity of the blue histograms. D) Cell surface expression staining of Grp94 clients as a function of virally transduced Grp94 construct. Cells were gated for EGFP expression as in C). Red = isotype control antibody histogram, blue = anti-client antibody histogram. The mean fluorescence intensity of the blue histogram is indicated.
