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Results: 6

1.
Fig. 2.

Fig. 2. From: Quantitative fluorescence imaging reveals point of release for lipoproteins during LDLR-dependent uptake.

LDL dissociates from the LDLR and enters EEA1-positive endosomes faster than β-VLDL. Surface LDLRs were saturated with Alexa546-LDL or β-VLDL at 4°C, washed, and shifted to 37°C for the indicated times. At each time point, cells were rapidly chilled to 4°C, fixed, and immunostained for LDLR and EEA1. Thirty fields from three separate experiments were processed for MC and PC of LDLR with LDL or β-VLDL (A and B) or for MC and PC of EEA1 with LDL or β-VLDL (C and D). Data are means ± SEM, n = 30.

Shanica Pompey, et al. J Lipid Res. 2013 March;54(3):744-753.
2.
Fig. 1.

Fig. 1. From: Quantitative fluorescence imaging reveals point of release for lipoproteins during LDLR-dependent uptake.

Quantitative imaging of clathrin, LDLR, and lipoprotein prior to uptake. Cells were treated or not with saturating concentrations of Alexa546-labeled LDL or β-VLDL at 4°C, washed, fixed, and counterstained for LDLR and clathrin by indirect immunofluorescence. A: MC and PC for LDLR-lipoprotein colocalization. Fifteen fields from three separate experiments were processed for MC of LDL with LDLR and β-VLDL with LDLR (MC LwR); MC of LDLR with LDL and LDLR with β-VLDL (MC RwL); and PC of LDLR with LDL and LDLR with β-VLDL (PC). Data are means ± SEM, n = 15. *P < 0.05 for β-VLDL compared with LDL. B: MC and PC for LDLR-clathrin colocalization. Fifteen fields from three experiments were processed for MC of LDLR with clathrin (MC RwC); MC of clathrin with LDLR (MC CwR); and PC of LDLR with clathrin in the presence or absence of LDL or β-VLDL. Data are means ± SEM, n = 15. *P < 0.05 compared with no lipoprotein.

Shanica Pompey, et al. J Lipid Res. 2013 March;54(3):744-753.
3.
Fig. 4.

Fig. 4. From: Quantitative fluorescence imaging reveals point of release for lipoproteins during LDLR-dependent uptake.

Deletion of the BC region slows both release and entry of lipoprotein into early endosomes. WT LDLR cells and LDLR-ΔBC cells were incubated with saturating concentration of Alexa546 LDL or β-VLDL at 4°C, washed, and shifted to 37°C for the indicated times. At each time point, cells were rapidly chilled to 4°C, washed, fixed and immunostained for LDLR and EEA1. Thirty fields from three experiments were used to generate MC and PC for colocalization. A and B: PC for the indicated receptors with LDL (A) or β-VLDL (B). C and D: PC for the EEA1 with LDL (C) or β-VLDL (D) in the indicated cells. E and F: PC for EEA1 with the indicated receptor in the presence of either LDL (E) or β-VLDL (F). G and H: MC for EEA1 with WT-LDLR or LDLR-ΔBC in the presence of either LDL (G) or β-VLDL (H). Data are means ± SEM, n = 30.

Shanica Pompey, et al. J Lipid Res. 2013 March;54(3):744-753.
4.
Fig. 6.

Fig. 6. From: Quantitative fluorescence imaging reveals point of release for lipoproteins during LDLR-dependent uptake.

Co-internalization of LDL and β-VLDL reveals that LDL and β-VLDL use common endocytic compartments. WT LDLR cells were incubated with 10 μg/ml Alexa488-LDL, 5 μg/ml Alexa546-β-VLDL, or a mixture of 10 μg/ml Alexa488-LDL and 1.25 μg/ml Alexa546-β-VLDL at 4°C, and then washed and incubated at 37°C for the indicated times. The 10:1.25 ratio resulted in a ∼50:50 ratio of LDL and β-VLDL binding. At each time point, cells were rapidly chilled to 4°C, washed, fixed and immunostained for either EEA1 (A and C) or LDLR (B). A: MC and PC for colocalization of LDL with β-VLDL for cells incubated with a mixture of the two lipoproteins. B: PC for LDLR with LDL alone, with LDL in the presence of β-VLDL, with β-VLDL alone, or with β-VLDL in the presence of LDL. C: PC for EEA1 with LDL alone, with LDL in the presence of β-VLDL, with β-VLDL alone, or with β-VLDL in the presence of LDL. D: Degradation assays in which 125I-LDL or β-VLDL were incubated with WT LDLR cells at 4°C in the absence or presence of sufficient unlabeled β-VLDL or LDL, respectively, to decrease 125I-lipoprotein binding in half. +β-VLDL and +LDL indicate assays in which unlabeled β-VLDL or LDL were present. Concentrations of unlabeled lipoprotein were titrated to reduce radiolabeled lipoprotein binding by 50%. Cells were then washed and incubated at 37°C for the indicated times and assayed for 125I-degradation products. Data was normalized to initially bound 125I-lipoprotein and are means ± SD, n = 4.

Shanica Pompey, et al. J Lipid Res. 2013 March;54(3):744-753.
5.
Fig. 5.

Fig. 5. From: Quantitative fluorescence imaging reveals point of release for lipoproteins during LDLR-dependent uptake.

Deletion of the BC region increases LDL retro-endocytosis. The indicated cells were assayed for LDL and β-VLDL accumulation (A and B), total fluorescence intensity (C and D), or degradation and excretion of lipoprotein (E and F). A and B: Cells were incubated with saturating concentrations of Alexa546-LDL or β-VLDL for the indicated times at 37°C, washed, fixed and processed by flow cytometry. Data was normalized to cellular fluorescence of WT cells following 4 h of uptake. Data are means ± SD from three independent experiments. C and D: Total integrated fluorescence from each experiment described in Fig. 4 was quantified. Data are means ± SD, n = 3. E and F: Cells were incubated with saturating concentrations of 125I-LDL or 125I-β-VLDL at 4°C, washed, and either assayed for surface-bound lipoprotein or incubated at 37°C for 4 h. Media from cells incubated at 37°C were assayed for degradation products of LDL (TCA soluble counts) or excreted LDL (TCA insoluble counts). Data are shown as a fraction of initially surface-bound lipoprotein and are means ± SEM, n = 9. *P < 0.05 for LDLR-ΔBC compared with WT LDLR.

Shanica Pompey, et al. J Lipid Res. 2013 March;54(3):744-753.
6.
Fig. 3.

Fig. 3. From: Quantitative fluorescence imaging reveals point of release for lipoproteins during LDLR-dependent uptake.

Lipoprotein release occurs prior to lipoprotein entry into early endosomes. A: LDL and β-VLDL internalize with similar kinetics. Surface LDLRs were saturated with 125I-LDL or 125I-β-VLDL at 4°C and then shifted to 37°C in the presence of 125I-lipoprotein for the indicated times. Surface-bound lipoprotein was then released by protease K, and surface and cell-associated (internal) lipoprotein was separated by centrifugation. Data are means ± SD, n = 4. B: Rates derived from linear regression of the data in (A). C and D: Lipoprotein internalization does not chase LDLRs into early endosomes. Data from Fig. 2 were processed for MC and PC of LDLR with EEA1 in the presence of LDL or β-VLDL. Data are means ± SEM, n = 30. E and F: Lipoprotein releases from the LDLR prior to entry of lipoprotein into early endosomes. Lipoprotein isosurfaces from three pulse-chase experiments were generated as described in Materials and Methods and in supplementary Fig. II. Isosurfaces were classified into four bins: surfaces that lack both LDLR and EEA1; surfaces that contain LDLR but not EEA1; surfaces that contain both LDLR and EEA1; and surfaces that contain EEA1 but not LDLR. Percentages of total lipoprotein surfaces for each bin were calculated for each experiment and are reported as means ± SEM, n = 3.

Shanica Pompey, et al. J Lipid Res. 2013 March;54(3):744-753.

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