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Fig. 7 | BMC Cell Biology

Fig. 7

From: Adopting the rapamycin trapping assay to track the trafficking of murine MHC class I alleles, H-2Kb

Fig. 7

Live cell imaging for Venus-FRB-Kb cycling. (a) Six different snapshots of live Jurkat cells coexpressing Venus-FRB-Kb and Cerulean-FKBP-Ii (double-transfected red and green cell = RG) or expressing only one of the constructs (only Ii in red = R cell) were taken from a 45 min movie (images were captured at a 2-min interval; snapshots are revealed at different allocated time points). Each image consists of four compiled z-stacks, where each ∆z = 1 μm. Before recording the movie, cells were transferred to a 37 °C heat stage in CO2-independent buffer and incubated first with cycloheximide (10 μg/ml) for 10 min and then with rapamycin (1 μM). Pearson’s coefficient was calculated from each image by defining a circular region of interest (ROI) around the Golgi area where the green fluorescence intensity of Venus-FRB-Kb accumulates (an arrow pointing to the circle in the first panel). For the cell only expressing Cerulean-FKBP-Ii in red (R cell), the ROI was drawn around the entire cell. (b) The graph shows the time-dependent change in Pearson’s coefficient based on the variation of the green fluorescence intensity with respect to the red fluorescence in the chosen ROI. There is an increase in Pearson’s coefficient for the RG but not for the control (R) cell. (c) There is a 25 % increase in the percentage of cells that show Venus-FRB-Kb localization in the ER upon addition of rapamycin (*p < 0.02). The error bars correspond to the SEM values from 2 sets of experiments (n > 250)

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