FIGURE 2:

Dual pulse-chase strategy for following simultaneous trafficking of Na,K-ATPase and E-cadherin. (A) Live SNAP-CLIP cells were preincubated with nonfluorescent blocking reagents (SNAP + CLIP block) or mock treated (No Block) for 30 min at 37°C, fixed, stained as described in Figure 1, and processed for immunofluorescence with an anti–myosin 1c antibody to label the lateral plasma membrane (blue). Bar, 10 μm. (B) The dual pulse-chase strategy to specifically detect biosynthetic trafficking. (C) SNAP-CLIP cells pretreated with blockers were washed and allowed to synthesize new protein for 30 min at 37°C (pulse). After the pulse period, samples were treated with CHX and incubated at 37°C for the indicated times before fixation and labeling as described. Bar, 5 μm. (D) The fraction of protein present at the plasma membrane (as defined by staining for myo-1C) in the experiments outlined in C as assessed with Manders colocalization analysis. Data are mean ± SD from three independent experiments.