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. 2021 Oct 31;18(6):1416–1432. doi: 10.1080/15548627.2021.1987672

Figure 5.

Figure 5.

FAIM regulates the tetramer formation of GAC. (A) A549 cells were transfected with empty vector or pcDNA3.1-HA-FAIM and mitochondria were isolated and glutaminase activity was measured. Data represent the average of three independent experiments (mean ± SD). **, P < 0.01. (B) A549 cells were transfected with control siRNA or FAIM siRNAs. Forty-eight h later, mitochondria were isolated and glutaminase activity was measured. Data represent the average of three independent experiments (mean ± SD). *, P < 0.05. (C and D) Mitochondria were isolated from A549 (C) and H1299 cells (D). The expression of mitochondria and cytosolic proteins were detected by western blot. (E) A549 cells were transfected with empty vector or pcDNA3.1-HA-FAIM and immunoprecipitation was performed followed by western blot. (F) A549 cells were transfected with indicated plasmids. The cells were lysed and analyzed using 4–16% Native PAGE, followed by immunoblot analysis. (G) A549 cells were transfected with the indicated siRNAs. The cells were lysed and analyzed using 4–16% Native PAGE, followed by immunoblot analysis. (H) A549 control cells and FAIM knockdown stable cell lines were lysed and analyzed using 4–16% Native PAGE, followed by immunoblot analysis. (I) A549 cells were transfected with pcDNA3.1-V5-GAC (WT GAC) or pcDNA3.1-V5-GAC mutants (GACS314A, GACS314D), then analyzed using 4–16% Native PAGE, followed by immunoblot analysis. (J) A549 cells were transfected with the indicated plasmids. The cells were lysed and analyzed using 4–16% Native PAGE, followed by immunoblot analysis. (K) A549 cells were co-transfected with the indicated plasmid and siRNAs. The cells were lysed and analyzed using 4–16% Native PAGE, followed by immunoblot analysis. (Figure F-K: GAC-T: GAC tetramer; GAC-M: GAC monomer). (L) A549 cells were co-transfected with pcDNA3.1-V5-GAC and the indicated siRNAs. Forty-eight h later, the cells were lysed and immunoprecipitation was performed followed by western blot. (M) A549 cells were transfected with indicated plasmids. Forty-eight h later, the cells were lysed and immunoprecipitation was performed. The expression of the indicated proteins was examined by western blot.