Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2019 Jan 1;30(1):69–81. doi: 10.1091/mbc.E18-01-0001

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2019 Chiang et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License.

PMC Copyright notice

FIGURE 7: — Slit2 affects Arl4A-induced Cdc42 activation and cell migration via regulating the Robo1-Arl4A and Robo1-srGAP1 associations. (A) Representative images of HEK293T cells transfected with the indicated plasmids and siRNA and then subjected to the Transwell assay. Purified Slit2 (0.2 µg/ml) was added to the lower chamber of the Transwell apparatus. The number of migrated cells in a field was calculated using ImageJ software after 22 h of migration. Histograms: Migration assay data were quantified based on three biological replicates. (B) Total protein (20 μg) was loaded onto a 10-well gel to detect proteins. Immunoblotting analyses were used to evaluate protein expression levels in cells transfected with the indicated plasmids and siRNA. The percentage of Robo1 after siRNA treatment was 11.5 ± 0.75%. (C) HEK293T cells were transiently transfected with the indicated plasmid and treated with 0.2 or 0.6 µg/ml purified Slit2. The cells were lysed and subjected to Cdc42 activity pull-down assays. To confirm the initial expression level, 2.5% of the total cell lysate (input) was loaded. Equal amounts of GST-tagged beads and cell lysates were used in each experiment as shown by Coomassie Blue staining. Histogram: Active Cdc42 was quantified based on three biological replicates. (D, E) HEK293T cells were transiently transfected with the indicated plasmids and then treated with purified Slit2 (µg/ml). Interactions were verified by in vivo coimmunoprecipitation with anti-Flag M2 magnetic beads. To confirm the initial expression level, 7.5% and 2.5% of the total cell lysates (input) were loaded. The heavy chain was used to ensure that equal amounts of M2 magnetic beads were loaded in each group. SDS–PAGE analysis was used to verify srGAP1, Robo1, and Arl4A expression and total cell lysates. Histograms: Co-IP assay data were quantified based on three biological replicates. The solid bars represent the mean ± SD. *, P < 0.01; **, P < 0.005; ***, P < 0.001 (A, C, and E: one-way ANOVA with Dunnett's post hoc multiple comparison test; D: two-tailed Student's t test).