QuikChange II XL Site-Directed Mutagenesis Kit, 10 Rxn

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- Use only the Dpn I enzyme provided; do not substitute with an enzyme from another source.

Protocol tips
- Use only the Dpn I enzyme provided; do not substitute with an enzyme from another source.

Publication protocol

Reagents
PDHA1 cDNA image clone (Open Biosystems) was used to engineer several PDHA1 variants with a FLAG epitope tag and were subsequently subcloned into pDEST27 for GST-tagged PDHA1 expression and purification in mammalian cells and pET53 vectors (Invitrogen) for His-tagged PDHA1 expression and purification in bacteria, respectively. Point mutations were introduced using QuikChange-XL site-directed mutagenesis kit (Stratagene). [5-3H]glucose, [1-14C]pyruvate and [2-14C]pyruvate were purchased from PerkinElmer Life Sciences. Stable knockdown of endogenous PDHA1 was achieved using a lentiviral vector harboring an shRNA construct (Open Biosystems; 5′-CGAATGGAGTTGAAAGCAGAT-3′). PDHA1 rescue H1299 cell lines were generated as described previously (11). Briefly, retroviral vector pLHCX (Clontech) containing shRNA-resistant, FLAG-tagged human PDHA1 WT or mutant forms harboring silent mutations in the shRNA-targeted region were transfected into H1299 cells containing shRNA directed against endogenous PDHA1. Antibody against PDHA1 was purchased from Invitrogen. Phospho-PDHA1 (Ser-293) antibody was purchased from Calbiochem. Phosphotyrosine antibody Tyr(P)-99 was purchased from Santa Cruz Biotechnology. Anti-FLAG, β-actin, and GST antibodies were purchased from Sigma. Specific antibody against phospho-PDHA1 (Tyr(P)-301) and acetyl-PDHA1 (K321-Ac) was generated by Cell Signaling Technology specifically for the current project and is not available commercially.

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Manufacturer protocol

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