FIGURE 8.

GFP-tagged TMEM184A colocalizes with rhodamine-heparin. A, A7r5 cells electroporated with GFP-TMEM184A as in Fig. 5 were treated with 100 μg/ml rhodamine-heparin for the indicated times, and then they were fixed with 4% PFA. Images are representative of two separate experiments. Scale bars = 10 μm. B, A7r5 cells and stable knockdowns for TMEM184A were treated with 100 μg/ml rhodamine-heparin for 10 min and fixed with 4% PFA. Images are representative of duplicates from two separate experiments. Scale bars = 10 μm. C, GFP-TMEM184A-transfected A7r5 cells were incubated with 200 μg/ml rhodamine-heparin, and cells were imaged immediately without fixing. Time-lapse confocal microscopy, initiated at about 4 min after heparin addition, identified colocalization of rhodamine-heparin (white vertical line) with a cluster of the GFP-TMEM 184A construct (scan zoom, 6.9; objective, ×63/1.4 oil differential interference contrast). The reference white bar points to the initial location and is a reference for concurrent movement of both labels. D, A7r5 cells and stable knockdowns for TMEM184A (KD) were either transfected with the GFP-TMEM184A construct or not transfected and treated as in B. At least 50 cells/condition (in each of three experiments) were analyzed for heparin uptake. **, p < 0.0001.