|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
| ||||||||||||||||||||
Files in this Data Supplement:
Fig. S1. The absence of Hip1r expression in Hip1r-null cells was confirmed by immunoblotting whole-cell lysates of wild-type cells (WT) and Hip1r-mutant cells (Hip1r-). The polyclonal serum against Hip1r is specific for Hip1r. Hip1r (A) polyclonal serum against Hip1r detects Hip1r in whole-cell lysates of WT but not Hip1r-mutant cells. Nonspecific bands are seen in both strains. (B) Affinity purification of the polyclonal serum against Hip1r eliminates nonspecific background bands seen in whole-cell lysates. (C-G) The polyclonal serum against Hip1r recognizes endogenous Hip1r in fixed cells. (A-D) WT and Hip1r-mutant cells were immunostained with affinity-purified serum against Hip1r. Wild-type cells display membrane and cytoplasmic puncta, whereas the Hip1r-mutant cells do not exhibit staining. (F-G) WT cells stained with the secondary antibody alone also do not exhibit staining.
Fig. S2. Growing Hip1r-null cells complete cytokinesis and are not deficient in pinocytosis or secretion. (A) Hip1r-null cells are able to complete cytokinesis, as measured by growth in suspension cultures. Wild-type (closed diamonds) and Hip1r-null cells (open circles) were grown in suspension. Clathrin-null cells (closed squares), which are deficient in cytokinesis in suspension cultures, were included as a control. (B) Hip1r-null cells are not deficient in pinocytosis. Wild-type (closed diamonds) and Hip1r-null cells (open circles) were incubated with FITC-labeled dextran (2 mg/ml). At the indicated time points, samples were collected and the amount of internalized FITC-labeled dextran was determined with a fluorometer. (C) Hip1r-null cells are not deficient in recycling an endocytosed marker. Wild-type (closed diamonds) and Hip1r-null cells (open circles) were loaded with FITC-labeled dextran (2 mg/ml) for 2 hours to fill their endocytic compartments. Cells were then incubated in media and, at the indicated time points, samples were collected, washed and the amount of internalized FITC-dextran remaining in the cells was determined with a fluorometer.
Fig. S3. Hip1r-null cells display normal clathrin and cortical actin. (A,B) Wild-type (WT) and Hip1r-null (Hip1r-) cells were fixed and stained with antibodies against clathrin. Both WT and Hip1r-null cells show fluorescent puncta at the plasma membrane and juxtanuclear staining. (C,D) To visualize F-actin, wild-type and Hip1r-null cells were stained with rhodamine-conjugated phalloidin. Both WT and Hip1r-null cells show prominent staining of F-actin at the cellular cortex. Bar, 10 μm.
| ||||||||||||||||||||
