Block it adenoviral rnai expression system

siRNAs were obtained from Gene Pharma (Shanghai, China). Specifically, H9C2 cells and primary cardiomyocytes were transfected with 100 nM of siRNA with lipofectamine RNAi MAX (Invitrogen, Waltham, MA, USA, 13778150) for 48 h. Adenoviruses encoding shRNAs were generated using BLOCK-iT Adenoviral RNAi Expression System (Invitrogen, K494100) according to the manufacturer’s protocol.
The following siRNAs and shRNA were used in this study:
Vps4a siRNA: GCUACUCAGGAGCAGAUAUTT,
Control siRNA: UUCUCCGAACGUGUCACGUTT,
Vps4a shRNA: GCTACTCAGGAGCAGATATTT,
Control shRNA: TTCTCCGAACGTGTCACGTTT.

The BLOCK-iT adenoviral RNAi expression system (Invitrogen) was used to construct adenoviral shRNAs for γ1, γ2, and γ3 vectors as previously described^{27 (link),39 (link),40 (link)}. The following sequences were used to generate shRNAs to effectively deplete γ subunit isoforms: γ1-2 (5′-GGTGGACATCTACTCCAAGTT-3′), γ1-3 (5′-CATCGGTCCCACTACT TTGA-3′); γ2-1 (5′-GCGTTTATATGCGATTCATGA-3′), γ2-2 (5′-GCAGGAGAACTTGAAC AAAGT-3′); γ3-1 (5′-CCCTCATCAAGAACCGAATC-3′), γ3-2 (5′-GGGCCTGAAATGCT TGGTTTC-3′). Subsequently, the vector of adenoviral shRNA for γ1 (γ1-3) was employed to generate AAV-vector. Regions in the pENTR/U6 vector containing the U6 promoter, Pol III terminator, and γ1-3 shRNA oligo or scrambled shRNA oligo were amplified by PCR and cloned into the AAV-BASIC vector (Vector Biolabs); these vectors were used to make AAV8 shRNAs for γ1 and scrambled shRNA. The adenoviral expression vectors of AMPKα1, α2, and β1 were generated as we reported previously¹⁵ . To generate the AMPKγ1 mutants, FLAG-tagged γ1-WT, and γ1 mutants with deletion of individual CBS domains were subcloned into the pENTR2B vector (Invitrogen) and transferred into the pAd/CMV/V5-DEST vector (Invitrogen) by recombination to generate adenoviral expression clones^{27 (link)}.