Skip to main content
. 2021 Sep 2;40(21):e108028. doi: 10.15252/embj.2021108028

Figure 2. CARS is critical for AMPK activation under cystine deprivation conditions.

Figure 2

  1. Venn diagram showing the overlap of CaMKK2‐binding proteins during cystine deprivation and cyst(e)ine metabolism‐related proteins. The schematic diagram shows the peptides detected in the results of IP‐MS assays of the AHCY and CARS proteins. The CaMKK2‐binding proteins are listed in Table EV3, and the cyst(e)ine metabolism‐related proteins are listed in Table EV4.
  2. 293T cells were transfected with HA‐CaMKK2 alone or with Flag‐CARS for 48 h and then cultured with cystine‐deficient medium or complete medium for 8 h. Cell lysates were immunoprecipitated with anti‐Flag, and a WB analysis was performed.
  3. 293T cells cultured with cystine‐deficient medium or complete medium for 8 h were harvested and subjected to immunoprecipitation with anti‐CaMKK2, followed by WB analysis with anti‐CaMKK2 and anti‐CARS.
  4. Flag pull‐down assays were performed with Flag‐EV or Flag‐fused CaMKK2 protein and His‐CARS purified from E. coli.
  5. WB analysis of p‐AMPK, p‐ACC, p‐Raptor and total AMPK ACC, Raptor protein expression in 293T cells transfected with shRNAs targeting CARS that were further treated with cystine‐deficient medium for 8 h. Actin served as the loading control.
  6. 293T cells were first transfected with shRNAs targeting CARS or NTC, then transfected with HA‐AMPKα1 or HA‐AMPKα1 and Flag‐CaMKK2 for 48 h, and then, they were cultured with cystine‐deficient medium for 8 h. Cell lysates were immunoprecipitated with anti‐Flag and subjected to Western blot analysis.
  7. 293T cells were transfected with shRNAs targeting CARS or NTC; then, they were cultured with cystine‐deficient medium for 8 h. Cell lysates were immunoprecipitated with anti‐AMPKα and subjected to WB analysis with anti‐AMPKα and anti‐CaMKK2.
  8. WB analysis of p‐AMPK, p‐ACC, p‐Raptor and total AMPK, ACC, Raptor, CARS protein expression in 293T cells and RCC4 cells overexpressing Flag‐CARS. Actin served as the loading control.
  9. WB analysis of p‐AMPK and total AMPK protein expression in CARS‐overexpressing 293T cells were further transfected with shRNAs targeting LKB1, CaMKK2 or TAK1. Actin served as the loading control.
  10. WB analysis of p‐AMPK and total AMPK protein expression in CARS‐overexpressing 293T cells and RCC4 cells that were treated with 1 µg/ml STO‐609 or DMSO for 8 h. Actin served as the loading control.

Source data are available online for this figure.