Mast Cell-specific Gangliosides and FcRI Follow the Same Endocytic Pathway From Lipid Rafts in RBL-2H3 Cells

Supplemental Material - Oliver et al. - Vol. 55, pp 315-325

Online Supplemental data for Oliver et al.: Figures.

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• Oliver et al., Supplemental Figure 1 - mAb BC4 is internalized and with time traffics to the dense lysosome fraction. RBL-2H3 cells were pulsed with I125-BC4 for 10 min and chased for 0 min, 10 min, 30 min, 1 hr, 2 hr or 3 hr at 37C. Cell fractions were prepared using Percoll density gradients. AcPase was used to mark the light membrane and dense lysosome fractions. The light membrane fraction consists of Golgi saccules, intracellular membranes such as endoplasmic reticulum and vesicles, plasma membrane and light lysosomes, while the dense lysosome fraction contains mainly dense lysosomes and mitochrondria (Oliver et al. 1989). I125-BC4 binds the α-subunit of FcεRI and activates the cells. At the start of the chase period (0 min) the majority of FcεRI is localized in the light membrane fraction of the density gradients (A). After 10 min chase, little I125-BC4 has moved into the dense lysosome fractions (B). However, at 30 min of chase some I125-BC4 is now present in the dense lysosome fraction. The amount of I125-BC4 in the dense lysosome fraction is increased at 1 hr (D) and 2 hr (E). By 3 hr of chase a significant amount of FcεRI can be found in the dense lysosome fraction (F). A representative experiment from 11 experiments is shown.
• Oliver et al., Supplemental Figure 2 - In unstimulated RBL-2H3 cells mAb AA4 remains associated with the light membrane fraction. RBL-2H3 cells were pulsed with I125-AA4 for 10 min and chased for 0 min, 10 min, 30 min, 1 hr, 2 hr or 3 hr. Cell fractions were prepared using Percoll density gradients. AcPase was used to mark the light membrane and dense lysosome fractions. At the start of the chase period (0 min) the majority of the I125-AA4 labeled gangliosides are localized in the light membrane fraction of the density gradients (A). After 10 min chase, almost no I125-AA4 has moved into the dense lysosome fraction (B), but by 30 min of chase some I125-AA4 is now present in the dense lysosome fraction. The amount of I125-AA4 in the dense lysosome fraction only slightly increased at 1 hr (D), 2 hr (E) and 3 hr of chase (F). A representative experiment from 11 experiments is shown.
• Oliver et al., Supplemental Figure 3 - In cells stimulated via FcεRI, mAb AA4 shows the same kinetics of internalization and trafficking as mAb BC4. RBL-2H3 cells were stimulated with unlabeled mAb BC4 and pulsed with I125-AA4 for 10 min and chased for 0 min, 10 min, 30 min, 1 hr, 2 hr or 3 hr. Cell fractions were prepared using Percoll density gradients. AcPase was used to mark the light membrane and dense lysosome fractions. At the start of the chase period (0 min) the majority of the I125-AA4 labeled gangliosides are localized in the light membrane fraction of the density gradient (A). After 10 min chase, virtually no I125-AA4 has moved into the dense lysosome fraction (B), but by 30 min of chase some I125-AA4 is now present in the dense lysosome fraction (C). The amount of I125-AA4 in the dense lysosome fraction increases at 1 hr (D) and 2 hr (E). By 3 hr of chase a significant amount of I125-AA4 can be found in the dense lysosome fraction (F). A representative experiment from 11 experiments is shown.