The pRK7-S6K1-WT plasmid that encodes the wild-type form of p70S6K and its control vector pRK7 were described previously and kindly provided by Dr. John Blenis (Harvard Medical School).27 (link) The HA-GSK3β-WT and HA- GSK3β-S9A plasmids that encode the wild-type and constitutively active form of GSK3β were described previously and purchased from Addgene, Inc. pcDNA plasmids were used as control. Transfection was conducted as described previously.20 (link)
Ha gsk3β s9a
Manufactured by Addgene
Sourced in United States
HA-GSK3β-S9A is a plasmid construct that expresses a mutant form of the human glycogen synthase kinase 3 beta (GSK3β) protein. The 'HA' prefix indicates that the protein is tagged with the hemagglutinin (HA) epitope, and the 'S9A' mutation introduces an alanine substitution at the serine 9 position of the GSK3β protein.
Automatically generated - may contain errors
3 protocols using ha gsk3β s9a
1
Overexpression of S6K1 and GSK3β
2
Modulating Mitochondrial Dynamics in SK-N-SH Cells
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Human SK-N-SH cells were cultured on 24-well culture plates at low density and incubated with DMEM with 10% FBS and 5.5 mmol/L d -glucose. Cells were then transfected with plasmids containing constitutively active forms of hemagglutinin–glycogen synthase kinase-3β (HA-GSK3β) (HA-GSK3βS9A) or a dominant-negative form of GSK3β (HA-GSKK85A) (Addgene), green fluorescence protein (GFP)-tagged wild-type Drp1 or Drp1K38A (provided by Dr. Yi-Ren Hong, Kaohsiung Medical University Hospital, Taiwan), or empty control vectors (GFP or HA) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Forty-eight hours after transfection, cells were treated with indicated reagents and assessed for changes in mitochondrial morphology and function as well as molecular signaling pathways.
3
Plasmids for Studying Protein Kinases and Deacetylases
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FLAG-UBR5 (#37188), HA-GSK3β (#14753), HA-GSK3β-S9A (#14754), and HA-GSK3β-K85A expression plasmids were from Addgene (USA). FLAG-AMPKα1 (CH805185), FLAG-SKP2 (CH896343), and FLAG-AKT1 (CH846646) were purchase from Vigenebio (Shangdong, China). SIRT7 and GSK3β deletion mutants were constructed into the p3× FLAG-CMV10 vector (Sigma). GST-tagged SIRT7 or SIRT7-S259A/Thr263A/Thr255A was constructed by cloning into pGEX-4T-3 (GE Healthcare). The other SIRT7 mutants were generated by the site-directed mutagenesis using the KOD-Plus-Neo DNA polymerase (TOYOBO). All plasmids were confirmed by DNA sequencing (Genewiz). Details of the primers used for the generation of these plasmids or other experiments are shown in Supplementary Table 2 .
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