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. 2018 Sep 25;475(18):2969–2983. doi: 10.1042/BCJ20180475

Figure 2. TBC1D1 binds AMPK-α1 and not AMPK-α2 heterotrimeric complexes.

Figure 2.

(A) Lysates from differentiated C2C12 myotubes lentivirally transduced with GFP or GFP-TBC1D1 subjected to GFP-trap and western blotting for AMPK-α isoforms. (B) Immobilised GST-His or GST-PTB1 + 2-His proteins were incubated with lysis buffer only (-) or lysates from Flp-In HEK293 cells stably expressing either FLAG-tagged AMPK-α1 or AMPK-α2. Washed complexes were analysed by western blotting. (C) Quantitation of data in (B), normalised to GST-PTB1 + 2-His and expressed in terms of FLAG-AMPK-α1 pull-down. Mean ± SEM; five independent experiments; two-tailed t-test *P < 0.05, **P < 0.01, ****P < 0.0001. (D) Immobilised GST or GST-PTB1 + 2 proteins were incubated with lysis buffer only (-) or purified recombinant AMPK αβγ heterotrimeric complexes as indicated and washed pull-downs analysed. Representative blots of four independent experiments. (E) Quantitation of data in (D), normalised to GST-PTB1 + 2. Mean ± SEM; four independent experiments; one-way ANOVA Dunnett's post-test *P < 0.05 ****P < 0.0001 cf pull-down of α1β1γ1.