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. 2012 Apr 1;23(7):1330–1342. doi: 10.1091/mbc.E11-09-0812

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© 2012 Mulkearns and Cooper. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 5: — Endocytic activity of enlarged CCSs. (A) Incubation at 4°C allows HA-mini-LDLR to cluster into enlarged plaques. Control or FCHO2 siRNA-treated HeLa-mini-LDLR cells were fixed, stained with antibody to HA, permeabilized, and then stained with antibody to Dab2. Images are adherent surface of cells. Scale bar: 10 μm. (aCHO2 is dispensable for LDLR endocytosis when cells are preincubated at 4°C for 1 h. HeLa mini-LDLR cells were incubated with antibody to HA for 1 h at 4°C, washed, warmed to 37°C for 2 min, then cooled, acid-stripped, and permeabilized, and HA-LDLR uptake was visualized using immunofluorescence. Values are mean fluorescence intensity ± SE of three experiments. Images are Z-stack projections. Scale bar: 20 μm. *, p < 0.05 by Student's t test. (C) TfnR fails to localize to enlarged CCSs with a 4°C incubation. Control or FCHO2 siRNA-treated HeLa-mini-LDLR cells were fixed, stained with antibody to TfnR, permeabilized, and then stained with antibody to Dab2. Images are adherent surface of cells. Scale bar: 10 μm. (D) Depletion of FCHO2 inhibits TfnR endocytosis even with a 4°C preincubation. HeLa-mini-LDLR cells were incubated with antibody to TfnR for 1 h at 4°C, washed, and moved to 37°C for 2 min to measure TfnR uptake. Values are mean fluorescence intensity ± SE of three experiments. Images are Z-stack projections. Scale bar: 20 μm. *, p < 0.05 by Student's t test. (E) Colocalization of LDLR and TfnR with Dab2 after 1 h at 4°C incubation. Using ImageJ, the percent of surface receptor area that colocalized with Dab2 area was measured, and mean and SEs were plotted. *, p < 0.05 by Student's t test. (F) Receptors are internalized from enlarged structures. HeLa-mini-LDLR cells were given antibody to HA-mini-LDLR for 1 h at 4°C and washed, and then were either fixed (1 h at 4°C) or warmed to 37°C for 2 min and fixed. Nonpermeabilized cells were stained with secondary antibody to surface HA-labeled receptor and then permeabilized and stained with anti-Dab2 and secondary antibodies for anti-Dab2 and internalized anti-HA–labeled receptor. Numbers on HA surface panels are mean fluorescence intensity ± SE of surface HA that colocalized with Dab2. Scale bar: 10 μm. *, p < 0.05 by Student's t test, compared with control after 1 h at 4°C.