Figure 6. Transport through the... | Rockefeller University Press

Figure 6.

$Figure 6. Transport through the endocytic pathway is required for FABP4 secretion. (A and B) Left: Adipocytes were incubated with 20 µM FSK in the presence or absence of 20 µM dynasore (A) or 60 µM dynasore (B), and at indicated times, medium fractions were collected and cells lysed. For each condition, performed in duplicate, 1% of total cell lysate and medium was analyzed by immunoblotting with anti-FABP4 and anti–α-tubulin antibodies. Right: Quantification of FABP4 secretion. For each condition, FABP4 secretion was calculated as a percentage of the signal detected in the medium compared with the total amount (the sum of FABP4 in both medium and lysate). Results are shown as the mean ± SD of three independent experiments. *, P < 0.05. (C–H) Left: Adipocytes were incubated in the presence of 20 µM FSK with 60 µM dynasore for 30 min, and cells were permeabilized with saponin before fixation. The colocalization of FABP4 with the indicated membrane markers was monitored by immunofluorescence microscopy. Bar, 10 µm. Pearson correlation coefficient (r) of colocalization, FABP4/M6PR r: 0.068 (C); FABP4/LAMP1 r: 0.079 (D); FABP4/EEA1 r: 0.427 (E); FABP4/CD63 r: 0.152 (F); FABP4/GRASP65 r: 0.029 (G); FABP4/GM130 r: 0.010 (H); n = 10 for each colocalization analysis. Right: Intensity profile graphs of FABP4 colocalization with the indicated membrane markers.$

Transport through the endocytic pathway is required for FABP4 secretion. (A and B) Left: Adipocytes were incubated with 20 µM FSK in the presence or absence of 20 µM dynasore (A) or 60 µM dynasore (B), and at indicated times, medium fractions were collected and cells lysed. For each condition, performed in duplicate, 1% of total cell lysate and medium was analyzed by immunoblotting with anti-FABP4 and anti–α-tubulin antibodies. Right: Quantification of FABP4 secretion. For each condition, FABP4 secretion was calculated as a percentage of the signal detected in the medium compared with the total amount (the sum of FABP4 in both medium and lysate). Results are shown as the mean ± SD of three independent experiments. *, P < 0.05. (C–H) Left: Adipocytes were incubated in the presence of 20 µM FSK with 60 µM dynasore for 30 min, and cells were permeabilized with saponin before fixation. The colocalization of FABP4 with the indicated membrane markers was monitored by immunofluorescence microscopy. Bar, 10 µm. Pearson correlation coefficient (r) of colocalization, FABP4/M6PR r: 0.068 (C); FABP4/LAMP1 r: 0.079 (D); FABP4/EEA1 r: 0.427 (E); FABP4/CD63 r: 0.152 (F); FABP4/GRASP65 r: 0.029 (G); FABP4/GM130 r: 0.010 (H); n = 10 for each colocalization analysis. Right: Intensity profile graphs of FABP4 colocalization with the indicated membrane markers.