Alpha4 is a non-canonical regulatory subunit of Type 2A protein phosphatases that interacts directly with the phosphatase catalytic subunits (PP2Ac, PP4c, and PP6c) and is upregulated in a variety of cancers. Alpha4 modulates phosphatase expression levels and activity, but the molecular mechanism of this regulation is unclear, and the extent to which the various Type 2A catalytic subunits associate with Alpha4 is also unknown. To determine the relative fractions of the Type 2A catalytic subunits associated with Alpha4, we conducted Alpha4 immunodepletion experiments in HEK293T cells and found that a significant fraction of total PP6c is associated with Alpha4, whereas a minimal fraction of total PP2Ac is associated with Alpha4. To facilitate studies of phosphatases in the presence of mutant or null Alpha4 alleles, we developed a facile and rapid method to simultaneously knockdown and rescue Alpha4 in tissue culture cells. This approach has the advantage that levels of endogenous Alpha4 are dramatically reduced by shRNA expression thereby simplifying interpretation of mutant phenotypes. We used this system to show that knockdown of Alpha4 preferentially impacts the expression of PP4c and PP6c compared to expression levels of PP2Ac. (C) 2016 Published by Elsevier Inc.