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

. 2020 Dec 22;220(1):e202002151. doi: 10.1083/jcb.202002151

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

© 2020 Chi et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

PMC Copyright notice

Figure 3. — NEDD1 facilitates initiation of cartwheel formation by directly recruiting SAS-6. (A) Total extracts of HEK293T cells expressing GFP-NEDD1 were immunoprecipitated with GFP-Trap beads and probed with antibodies against SAS-6, STIL, and PLK4. (B) Total extracts of HEK293T cells were immunoprecipitated with anti-NEDD1 antibody and probed with antibodies against SAS-6 and NEDD1. (C) In vitro SAS-6 and NEDD1 binding assay. GFP-Trap beads coupled with purified GFP-Strep or GFP-SAS-6-Strep were incubated with purified Strep-NEDD1 and analyzed using anti-NEDD1 antibody. The indicated protein loads are shown by Coomassie blue staining. (D) Total extracts of HEK293T cells cotransfected with Myc-NEDD1 and full-length or truncated GFP–SAS-6 proteins were immunoprecipitated with GFP-Trap beads and probed with antibodies against Myc and GFP. (E) Total extracts of HEK293T cells transfected with full-length or truncated GFP-NEDD1 were immunoprecipitated with GFP-Trap beads and probed with antibodies against SAS-6 and GFP. (F) Schematic of the interactions between NEDD1 and SAS-6. (G) HEK293T cells transfected with GFP-NEDD1 and synchronized at the G1/S transition were released into fresh medium for the indicated phases. The total cellular extracts were then immunoprecipitated with GFP-Trap beads and probed with antibodies against SAS-6, PLK4, and GFP. Cyclin B1 was used as a marker for the M phase. (H) U2OS cells treated with siRNA against SAS-6 or STIL were coimmunostained with antibodies against NEDD1 and SAS-6 or NEDD1 and STIL. DNA was stained with DAPI. Scale bars, 5 µm (large images) or 0.5 µm (inset images). (I) Comparisons of the relative fluorescence intensities of NEDD1 in H. Three independent replicates of >30 cells per replicate were quantified. Error bars indicate mean ± SD. ns, no significant difference (two-tailed t test). NC, negative control; WD repeat, Tryptophan-Aspartic acid repeat.