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 May 13;219(6):e20201405. doi: 10.1084/jem.20201405

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

This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

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. — Defective autophagy in T cells from Pt 1 and Gimap6^−/− mice. (A) Whole splenocytes from Gimap6^−/− (KO) or Gimap6^+/+ (WT control, Ctrl) mice were treated with 1 μg/ml each of anti-CD3/CD28 for 3 d and then with 10 nM Baf or vehicle (Veh) for 2 h. Flow cytometry of LC3 expression (LC3-II) in CD4⁺ and CD8⁺ T cells. (B) Quantification of A for the indicated days. **, P < 0.01 (unpaired Student’s t test). n_KO = 5; n_control = 8. (C) Autophagic flux of A, calculated as (gMFI LC3_Baf − gMFI LC3_Veh)/gMFI LC3_Veh. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (unpaired Student’s t test). n_KO = 5; n_control = 8. Shown is one of two independent repeats. gMFI, geometric MFI. (D) Flow cytometry histograms of LC3-II in human PBMCs from Pt 1 and healthy controls (Ctrl) stimulated with 1 μg/ml each of anti-CD3/CD28 for 3 d before treatment with Baf or vehicle (Veh) followed by flow cytometry and gating on CD4⁺ and CD8⁺ cells. (E) Quantification of gMFI of LC3-II of D as in B for the indicated days. (F) Quantification of autophagic flux of D as in C. Shown is one of two independent repeats and each dot indicates an individual donor in E and F. (G) 600 µM GABARAPL2 (GABA2) was titrated into 50 µM GIMAP6 (R134D; G6) and the resulting heat change was monitored in an ITC device. Data were fitted to a K_D of 40 + −10 nM (n = 0.63 + −0.01). Shown is one of three experiments. (H) GTPase inhibition of 2.5 μM GIMAP7 (G7) by various concentrations of a 1:1 M GIMAP6 (G6)-GABARAPL2 (GABA2) complex. Shown is one of three experiments. (I) WB of HEK293T cells transfected with empty vector (EV), HA-tagged GIMAP6^WT (WT-HA), or HA-tagged GIMAP6^G153V (G153V-HA) for 1 d. GIMAP6^WT and GIMAP6^G153V were immunoprecipitated with HA tag antibody. The cell lysate and IP were probed for GABARAPL2, HA, and HSP90. Shown is one of three experiments. (J) WB of Jurkat cells transduced with lentivirus of empty vector (EV), HA-tagged GIMAP6^WT (WT-HA), or HA-tagged GIMAP6^G153V (G153V-HA) and selected with puromycin (1 µg/ml) for 4 d. GIMAP6^WT and GIMAP6^G153V were immunoprecipitated with HA tag antibody. The cell lysate and IP were probed for GABARAPL2, GIMAP7, HA, and HSP90. Shown is one of three experiments. Bars (B, C, E, F, and H) represent mean ± SEM.