Figure 3. p62 is captured together with... | Rockefeller University Press

Figure 3.

$p62 is captured together with La into the lumen of late endosomes. (A) Mechanically ruptured MDA-MB-231 cells were subjected to differential centrifugation. Immunoblot analysis of the 5,000 × g pellet (P5K), 20,000 × g pellet (P20K), and 100,000 × g pellet (P100K) was conducted to evaluate the presence of the indicated proteins. (B) Quantification of the indicated proteins within the membrane pellet fractions from Fig. 3 A (n = 3). (C) Immunoblot analysis of proteinase K protection assays on a P20K membrane fraction to evaluate whether the indicated proteins were sequestered within the lumen of a detergent-sensitive compartment. (D) Quantification of the proteinase K protection experiments from Fig. 3 C (n = 3). (E) Airyscan microscopy of endogenous La, CD63, and p62 from MDA-MB-231 cells permeabilized with 0.02% saponin. Green: La; magenta: CD63; blue: p62. Scale bar: 10 µm. (F) Quantification of La, CD63, and p62 fluorescence intensities from point A to point B of the indicated inset of Fig. 3 E. (G) Airyscan microscopy of endogenous LC3, CD63, and p62 from MDA-MB-231 cells permeabilized with 0.02% saponin. Green: LC3; magenta: CD63; blue: p62. Scale bar: 10 µm. (H) Quantification of LC3, CD63, and p62 fluorescence intensities from point A to point B of the indicated inset of Fig. 3 G. Source data are available for this figure: SourceData F3.$

p62 is captured together with La into the lumen of late endosomes. (A) Mechanically ruptured MDA-MB-231 cells were subjected to differential centrifugation. Immunoblot analysis of the 5,000 × g pellet (P5K), 20,000 × g pellet (P20K), and 100,000 × g pellet (P100K) was conducted to evaluate the presence of the indicated proteins. (B) Quantification of the indicated proteins within the membrane pellet fractions from Fig. 3 A (n = 3). (C) Immunoblot analysis of proteinase K protection assays on a P20K membrane fraction to evaluate whether the indicated proteins were sequestered within the lumen of a detergent-sensitive compartment. (D) Quantification of the proteinase K protection experiments from Fig. 3 C (n = 3). (E) Airyscan microscopy of endogenous La, CD63, and p62 from MDA-MB-231 cells permeabilized with 0.02% saponin. Green: La; magenta: CD63; blue: p62. Scale bar: 10 µm. (F) Quantification of La, CD63, and p62 fluorescence intensities from point A to point B of the indicated inset of Fig. 3 E. (G) Airyscan microscopy of endogenous LC3, CD63, and p62 from MDA-MB-231 cells permeabilized with 0.02% saponin. Green: LC3; magenta: CD63; blue: p62. Scale bar: 10 µm. (H) Quantification of LC3, CD63, and p62 fluorescence intensities from point A to point B of the indicated inset of Fig. 3 G. Source data are available for this figure: SourceData F3.

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