Figure 6.

ABHD5 directly interacts with BECN1 to inhibit the cleavage of BECN1. (A) Reciprocal GST affinity isolation and immunoblotting of ABHD5 and BECN1 in wild-type CCD841CON cells. (B) GST-tagged ABHD5 was purified and incubated with increasing doses of purified BECN1 for in vitro detection of their direct association. (C) Immunoblotting of ABHD5 in the lysates of CCD841CON cells affinity isolated with GST-tagged full-length or different domains of BECN1 as indicated. The domain structure of BECN1 is given above the data. (D) In vitro binding assay with FLAG-tagged different domains of ABHD5 and His-tagged full-length BECN1 as indicated. The domain structure of ABHD5 is given above the data. (E) A close-up view of the ionic layer at the center of the crystal structure of the ABHD5-BECN1 complex is shown. (F) ABHD5-silenced and control cells were transfected with full-length ABHD5 or an ABHD5 mutant (mutated in the binding domain to BECN1) and subjected to EBSS treatment. The cytosolic and mitochondrial fractions were analyzed by western blotting using anti-BECN1-C with COX4I as a loading control, and the nuclear fractions were analyzed by western blotting using anti-BECN1-N with histone 2A as a loading control. (G, H) ABHD5-silenced and control CCD841CON cells were transfected with full-length ABHD5 or an ABHD5 mutant (mutated in the binding domain to BECN1) and subjected to EBSS treatment. Number of autolysosomes and cell viability were statistically analyzed.(*, p < 0.01; ns, no significance).