The GFP-CaMKII beta construct was obtained from Addgene (Addgene Cat #21227). The His-tagged, wild-type (WT) CRABP1 DNA construct for bacterial expression and subsequent protein purification was generated as described in Wook Park et al. (2019) (link). The Flag-tagged, WT CRABP1 DNA constructs for mammalian expression used in HEK293T CRABP1-CaMKII cell assays were described in Wook Park et al. (2019) (link). Constructs of alanine point mutants of His-tagged CRABP1 and Flag-tagged CRABP1 were generated using the Q5® Site-Directed Mutagenesis Kit (New England BioLabs Inc., Cat #E0554S) according to the manufacturer’s instructions. To validate successful site-directed mutagenesis, the relevant regions of the CRABP1 insert from each mutant construct were validated by Sanger sequencing, which was performed by the University of Minnesota Genomics Center Facility (Minneapolis, MN).
Chemical reagents utilized in this study are as follows: Tris-d11 solution (Sigma Cat # 486248), sodium acetate-d3 (Sigma Cat # 176079), dithiothreitol (DTT) (Gold Biotechnology Cat# DTT10), Tris (2-carboxyethyl)phosphine hydrochloride solution (Sigma Cat # 646547), dimethyl sulfoxide (DMSO) (Sigma Cat #D8418), and ionomycin salt (Sigma Cat #I0634). Ionomycin for molecular and cell studies was prepared by dissolving it in DMSO.
Chemical reagents utilized in this study are as follows: Tris-d11 solution (Sigma Cat # 486248), sodium acetate-d3 (Sigma Cat # 176079), dithiothreitol (DTT) (Gold Biotechnology Cat# DTT10), Tris (2-carboxyethyl)phosphine hydrochloride solution (Sigma Cat # 646547), dimethyl sulfoxide (DMSO) (Sigma Cat #D8418), and ionomycin salt (Sigma Cat #I0634). Ionomycin for molecular and cell studies was prepared by dissolving it in DMSO.
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