American Society of Hematology

Figure 2

$Figure 2. Galectin-5 secretion is associated with the endosomal pathway. Endosomal vesicles were prepared and loaded on a linear sucrose gradient, as described in “Methods.” (A) Fractions 1 to 9 were collected from the top of the gradient and after TCA/acetone precipitation, proteins were separated by SDS-PAGE and analyzed by Western blot for the presence of TfR and Gal-5. (B) Fractions were collected and densities (g/mL) were obtained by refractometry. Pooled fractions corresponding to cytosol (Cyt), multivesicular endosomes (MVE), and endocytic vesicles (EV) were processed by SDS-PAGE and analyzed by Western blot for the presence of TfR and Gal-5. (C) Endosomal vesicles (100 μg protein) were subjected to trypsin digestion (150 μg/mL for 1 hour at RT) in the absence or presence of Triton X-100 (1.5%), then loaded on SDS-PAGE gels and analyzed by Western blot for Gal-5. A nontrypsinized sample was used as control.$

Galectin-5 secretion is associated with the endosomal pathway. Endosomal vesicles were prepared and loaded on a linear sucrose gradient, as described in “Methods.” (A) Fractions 1 to 9 were collected from the top of the gradient and after TCA/acetone precipitation, proteins were separated by SDS-PAGE and analyzed by Western blot for the presence of TfR and Gal-5. (B) Fractions were collected and densities (g/mL) were obtained by refractometry. Pooled fractions corresponding to cytosol (Cyt), multivesicular endosomes (MVE), and endocytic vesicles (EV) were processed by SDS-PAGE and analyzed by Western blot for the presence of TfR and Gal-5. (C) Endosomal vesicles (100 μg protein) were subjected to trypsin digestion (150 μg/mL for 1 hour at RT) in the absence or presence of Triton X-100 (1.5%), then loaded on SDS-PAGE gels and analyzed by Western blot for Gal-5. A nontrypsinized sample was used as control.

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