Protein phosphatase 2A (PP2A) B ''-family subunits have Ca2+-binding EF-hand motifs and can bind PP2A substrates. Arabidopsis thaliana PP2A B ''-family subunits are encoded by six genes, and bind a transcription factor, VIP1. VIP1 is dephosphorylated and nuclear-localized by hypo-osmotic stress. However, whether PP2A B ''-family subunits mediate the VIP1 dephosphorylation is unclear. Here, we show by yeast two-hybrid and in vitro pull down assays that Arabidopsis PP2A B ''-family subunits bind Arabidopsis PP2A A (scaffold) subunits. We also show that VIP1 dephosphorylation in vitro can be induced by a PP2A B ''-family subunit. (C) 2020 Elsevier Inc. All rights reserved.