Figure 1: α-SNAP associates with AMPK bound to ATP. | Nature Communications

Figure 1: α-SNAP associates with AMPK bound to ATP.

From: α-SNAP inhibits AMPK signaling to reduce mitochondrial biogenesis and dephosphorylates Thr172 in AMPKα in vitro

Figure 1

(a) HEK293 cells as control (lane 1) and cells stably expressing FLAG-AMPKγ1 were treated for 20 min with vehicle (lane 2) or 2 μM oligomycin (oligo) (lane 3). AMPK was isolated by FLAG immunoprecipitation (IP) and proteins resolved by SDS–PAGE, followed by silver staining. The AMPK β and γ subunits (lane 2 and 3) are marked with arrows, and the prominent 35 kDa band (lane 2) was excised and analysed by LC/MS/MS mass spectrometry. (b) Immunoblots of cell extracts (upper panels) and immunoprecipitates (IP, lower panels) of endogenous α-SNAP, compared with non-specific IgG as a control. HEK293T cells were untreated as control or treated with 25 mM 2-deoxyglucose (2-DG), or 3 mM phenformin (phen). (c) FLAG-AMPKα2, β1, γ1 were co-expressed with HA-α-SNAP in HEK293T cells. Cell extracts and HA and FLAG immunoprecipitates were immunoblotted. (d) FLAG-AMPKγ1 wild-type (WT) and R299G were expressed in HEK293T cells and recovered on beads as FLAG immunoprecipitates. Beads were incubated with purified recombinant α-SNAP protein without or with addition of the indicated nucleotides. Samples were immunoblotted and staining intensity normalized relative to samples with no nucleotide added. Data represent means±s.e.m. for n=3 and statistical analysis of ANOVA followed by multiple Fisher’s test was performed (WT NONE versus ATP, ATP-γS and AMP-PNP: P<0.02; WT NONE versus AMP: P=0.997; R299G variant, P=0.927).

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