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 May 9;294(25):9844–9857. doi: 10.1074/jbc.RA119.007655

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

© 2019 Shang et al.

Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.

PMC Copyright notice

Figure 2. — USP9X deubiquitinates BCL9. A, HeLa cells with Dox-inducible expression of stably integrated FLAG–BCL9 were co-transfected with control siRNA or USP9X siRNAs together with HA–Ub as indicated. Cellular extracts were prepared for co-immunoprecipitation assays with anti-FLAG followed by IB with anti-HA. B, mass spectrometry analysis of BCL9 ubiquitin conjugation sites. HeLa cells stably expressing FLAG–BCL9 were co-transfected with HA–Ub. Cellular extracts were collected and sequentially purified with anti-FLAG affinity gel and HA affinity gel to enrich HA–Ub-conjugated BCL9. After trypsinization, the retrieved peptides were subjected to MS analysis. Fragmentation spectrums and parameters of the identified BCL9 peptides with the di-glycine remnants are shown. C, HeLa cells with Dox-inducible expression of stably integrated FLAG–BCL9/K23R, FLAG–BCL9/K212R, or FLAG–BCL9/K906R were co-transfected with control siRNA or USP9X siRNAs together with HA–Ub as indicated. Cellular extracts were prepared for co-immunoprecipitation assays with anti-FLAG followed by IB with anti-HA. D, HeLa cells with Dox-inducible expression of stably integrated FLAG–BCL9 were co-transfected with control siRNA or USP9X siRNAs together with HA–Ub/Lys-48–only or HA–Ub/Lys-63–only as indicated. Cellular extracts were prepared for co-immunoprecipitation assays with anti-FLAG followed by IB with anti-HA. E, HeLa cells with Dox-inducible expression of stably integrated FLAG–BCL9/K212R were co-transfected with control siRNA or USP9X siRNAs together with HA–Ub/Lys-63–only as indicated. Cellular extracts were prepared for co-immunoprecipitation assays with anti-FLAG followed by IB with anti-HA.