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

. 2022 Oct 20;12:17571. doi: 10.1038/s41598-022-22295-w

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

© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

PMC Copyright notice

Hakai regulates ubiquitination and degradation of FASN via lysosome. (a) Analysis of FASN degradation by cycloheximide chase. FASN half-life in HT29 cell line was determined by treatment with 10 µg/ml Cycloheximide for the indicated time course and compared with FASN half-life in Hakai-silenced HT29 cells compared to control HT29 cells. Hakai-silencing was performed by viral transduction induced with 1 µg/ml of Doxycycline for 72 h. Quantification of endogenous FASN protein half-life are shown (right panel). (b) Coimmunoprecipitation of Hakai and FASN. Hakai was immunoprecipitated in HCT-116 cells transfected with pcDNA-Flag-Hakai and v-Src. Hakai immunoprecipitation was performed using anti-Hakai antibody. Coimmunoprecipitation was evaluated by western blot as described in materials and methods using the indicated antibodies. GAPDH signal was used as protein loading control. (c) Hakai-dependent ubiquitination of FASN Flag-Hakai, v-Src and HA-ubiquitin were transiently transfected into HCT116 cells. Immunoprecipitation was performed with the anti-FASN antibody before Western blotting, using the indicated antibodies. (d) FASN and Hakai levels in HCT-116 cells treated with the lysosome degradation inhibitor Chloroquine, the autophagy inhibitor 3-Methyladenine and the proteasome inhibitor MG132 at the indicated concentration times described in materials and methods. LC3 I/II levels were analyzed as a positive control of chloroquine and 3-MA treatment and β-catenin for MG132 treatment. (e) HCT116 cells were transiently transfected with pcDNA-Flag-Hakai and the day after transfection cells were treated with chloroquine at 50 µM for 24 h. (f) Effect of Hakin-1 inhibitor on FASN expression. Hakin-1 was used at increasing concentrations for 48 h in HCT-116 cells. (g) Effect of Hakin-1 on Hakai-dependent ubiquitination of FASN. Flag-Hakai and HA-ubiquitin were transiently transfected into HCT116 cells. Immunoprecipitation was performed with the anti-FASN antibody before Western blotting using the indicated antibodies. GAPDH was used as protein loading control. The blots images were cropped prior hybridization and original blots are included in Supplementary Information.