Figure 2. Generation of β1β2 dKO cells.

Clones of HEK293T cells were isolated by flow cytometry following transfection with plasmids containing Cas9 and guide RNAs targeting the β1- and β2-isoforms of AMPK. (A) A representative capillary western blot of β subunit expression in five different clones compared with wild-type HEK293T is shown. Lower molecular mass cross-reacting bands, potentially due to truncated β-subunit protein, are indicated. (B) Total AMPKα and (C) AMPKγ1 expression in β1β2 dKO cells relative to wild-type cells were determined by western blotting. (D) Wild-type and β1β2 dKO cells were incubated with DMSO or 991 (1 μM) for 30 min. Levels of ACC and Thr¹⁷² phosphorylation, together with β-subunit expression, were determined. In each case, a representative blot with two independent samples is shown.