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

. 2023 Jun 2;9(22):eadg4993. doi: 10.1126/sciadv.adg4993

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

Copyright © 2023 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

PMC Copyright notice

Fig. 4. — (A) Interaction assay of Vps34 with Vps15, Beclin1, UVRAG, and Atg14L in LDHA knockdown HEK293T cells that rescued empty vector, LDHA^S196A-Flag, and LDHA^S196D-Flag. (B) Interaction assay of Vps34 lactylation sites mutants with Vps15, Beclin1, UVRAG, and Atg14L in HEK293T cells. Vps34^WT-Flag, Vps34^K356R-Flag, Vps34^K781R-Flag, and Vps34^2KR-Flag were transfected into HEK293T cells. (C) Interaction assay of Vps34 lactylation sites mutants with Vps15, Beclin1, UVRAG, and Atg14L in HEK293T cells in normal medium or EBSS medium for 3 hours. Interaction assay as in (B). (D) Interaction assay of Vps34 with Vps15, Beclin1, UVRAG, and Atg14L in HEK293T cells that were cultured in normal medium or normal medium containing lactate (1 mM) for 24 hours. Interaction assay as in (A). (E) Interaction assay of Vps34 with Vps15, Beclin1, UVRAG and Atg14L in TIP60 knockdown HEK293T cells that were cultured normal medium or EBSS medium for 3 hours. Interaction assay as in (A). (F) Vps34 kinase activity assay by GFP-FYVE2. GFP-FYVE2 was expressed in U2OS cells (control shRNA, Vps34 KD/rescued by vector, Vps34^WT, and Vps34^2KR) and analyzed by confocal microscopy. Scale bar, 10 μm. (G) Quantification of the relative GFP-FYVE2 puncta. Data are shown as means ± SD; **P < 0.01, n = 30. (H) GFP-DFCP1 puncta assay in U2OS cells (control shRNA, Vps34 KD, and Vps34 KD/rescued by Vps34^WT and Vps34r^2KR) under EBSS medium for 2 hours. Scale bar, 10 μm. (I) Quantification of the relative GFP-DFCP1 puncta. Data are shown as means ± SD; **P < 0.01, n = 30. (J) PI(3)P level determined by PI(3)P ELISA kit in U2OS cells (control shRNA and Vps34 KD/rescued by vector, Vps34^WT, and Vps34^2KR). **P < 0.01, n = 5. (K) Kinase activity of Vps34^WT and Vps34^2KR in vitro by PI(3)P ELISA kit. Vps34^WT and Vps34^2KR were purified from insect cells sf9, and TIP60-HA was purified from HEK293T cells. Vps34 lactylation was analyzed by Pan-Kla antibody. Data are shown as means ± SD; **P < 0.01, n = 3.