Figure 1.
CRT enhances ATZ trafficking in K42 mouse embryonic fibroblasts, and this effect is partially dependent on glycan binding.A, EM structure of CRT (PDB ID 6ENY) depicting the glycan-binding site which includes residue Tyr-92 within the globular domain. CRT's acidic domain is helical and the P-domain forms an extended β-hairpin structure. B, workflow for the estimation of media and cellular fluorescence in Fig. 1, Fig. 3, Fig. 4, Fig. S1, and Fig. S3. Briefly, media was collected 48 h post-transfection and cleared of debris by high-speed centrifugation. eYFP fluorescence was measured at an excitation wavelength of 514 nm and emission wavelength of 527 nm. Cellular fluorescence (post-fixation) was measured in the FITC channel on a flow cytometer. C, representative flow cytometry dot plots of cellular eYFP fluorescence in untransfected, eYFP-AAT–transfected, or eYFP-ATZ–transfected K42 CRT−/− and CRT WT or CRT Y92A cells. D, percentage live cells of all cells (pre-gated on forward and side scatter) and percentage of eYFP+ cells identified from the total live cell population. E, total media fluorescence and cell MFI values were normalized relative to corresponding values from CRT−/− cells in eYFP-AAT– or eYFP-ATZ–transfected cells. F, the ratio between the media and cell fluorescence values calculated as . For D–F, data were obtained from nine independent transfections of the indicated K42 cells and are shown as mean ± S.D. (error bars). Repeated measures (RM) one-way ANOVA analysis was performed for each set of measurements, comparing CRT−/−, CRT WT, and CRT Y92A. Because the data in panel E is normalized, for this panel, RM one-way ANOVA analysis was performed on the log-transformed data. Only significant comparisons are indicated. *p < 0.05, **p < 0.01, ***p < 0.001. See also Fig. S1 for additional replicates comparing K42 CRT−/− and CRT WT cells and Fig. S3, A–C for individual experimental trends of the eYFP-ATZ data.