Figure 1. Cytosolic La is captured into... | Rockefeller University Press

Figure 1.

$Cytosolic La is captured into the lumen of late endosomes. (A) Mechanically ruptured MDA-MB-231 cells were subjected to differential centrifugation. Immunoblot analysis of the 5,000 × g pellet (P5K), 20,000 × g pellet (P20K), and 100,000 × g pellet (P100K) was conducted to assess the presence of the indicated proteins. (B) Quantification of the indicated proteins within the membrane pellet fractions from Fig. 1 A (n = 3). (C) Immunoblot analysis of proteinase K protection assays conducted on a P20K membrane fraction to assess whether the indicated proteins were sequestered within the lumen of a detergent-sensitive compartment. (D) Quantification of the proteinase K protection experiments from Fig. 1 C (n = 3). (E) Airyscan microscopy of endogenous La with DAPI counterstain from MDA-MB-231 WT cells permeabilized with either 0.1% TX-100 or 0.02% saponin. Green: La; cyan: DAPI. Scale bar: 10 µm. (F) Airyscan microscopy of endogenous La and CD63 from MDA-MB-231 WT cells permeabilized with 0.02% saponin. Green: La; magenta: CD63. Scale bar: 10 µm. (G) Quantification of La and CD63 fluorescence intensity from point A to point B in the indicated inset of Fig. 1F. Source data are available for this figure: SourceData F1.$

Cytosolic La is captured 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 assess the presence of the indicated proteins. (B) Quantification of the indicated proteins within the membrane pellet fractions from Fig. 1 A (n = 3). (C) Immunoblot analysis of proteinase K protection assays conducted on a P20K membrane fraction to assess whether the indicated proteins were sequestered within the lumen of a detergent-sensitive compartment. (D) Quantification of the proteinase K protection experiments from Fig. 1 C (n = 3). (E) Airyscan microscopy of endogenous La with DAPI counterstain from MDA-MB-231 WT cells permeabilized with either 0.1% TX-100 or 0.02% saponin. Green: La; cyan: DAPI. Scale bar: 10 µm. (F) Airyscan microscopy of endogenous La and CD63 from MDA-MB-231 WT cells permeabilized with 0.02% saponin. Green: La; magenta: CD63. Scale bar: 10 µm. (G) Quantification of La and CD63 fluorescence intensity from point A to point B in the indicated inset of Fig. 1F. Source data are available for this figure: SourceData F1.

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