Fig. 3

Aberrant TF trafficking by CAMK4^−/− and DKO HEK293 cells. a-c: Live confocal fluorescent images showing localization of FITC-TF and RFP-TFRC in wild-type, CAMK4^−/− and DKO HEK293 cells. The cells were pulse-labeled with 25 μg FITC-TF for 1 h followed by a triple wash in 1xPBS buffer and then incubated in serum-free media. Following pulse-labeling, cells were imaged at 4 h and 24 h. The yellow arrows indicate membrane localization of RFP-TFRC and white arrows indicate internalized TF in vesicular structures. The white rectangle area indicates the region used for plot profiling using ImageJ software. Scale: 10 μm. d-i: Plot profiles of TF and RFP-TFRC intensities in the white rectangular areas marked in figure A-C respectively. Green arrows indicate the peaks representing membrane-localized RFP-TFRC/FITC-TF. The black dotted rectangular areas in Figs. E, G and J represent the peak areas used in determining the relative amount of TFRC/TF associated with the membranes. j: Scatter plot showing the relative amount of FITC-TF associated with membrane-bound RFP-TFRC after 24 h of pulse-labeling. N = 51, ~ 10 replicates from 5 independent experiments. P values by one-way ANOVA test followed by multiple comparisons.