FIGURE 2.

PP4R4, a novel PP4c interactor, is a ∼100-kDa protein resembling the PP2A A subunit. A, domain architecture similarities between human PP4R4, PP2A Aα, and PP4R1. HEAT repeats are indicated by black boxes. The percentage identity between PP4R4 and the other HEAT repeats over the shaded portion of the sequence is indicated. The PP2A Aα HEAT repeats responsible for interaction with the catalytic subunit and the B′γ subunit are indicated. aa, amino acids. B, chemically synthesized siRNAs directed against PP4R4 were transfected into HEK293 cells. 72 h after transfection, cells were collected. Equal amounts of lysate from each sample were separated by SDS-PAGE, and the proteins were transferred to nitrocellulose. Crude antiserum from a rabbit injected with glutathione S-transferase-PP4R4 C terminus was used for immunoblotting (IB). The arrow indicates the position of the ∼100-kDa band, which disappears after transfection of PP4R4 siRNAs. C, anti-PP4R4 was used in immunoblotting of lysate from untransfected cells (left) or cells stably expressing FLAG-PP4R4 (SDS-7.5%-PAGE). D, effect of silencing PP4c on the expression levels of PP4R4. U2OS cells transfected with PP4c (or PP4R4, two independent siRNAs) siRNAs were collected after 40 h and processed for immunoblotting analysis as in B using antibodies to PP4c or PP4R4. The position of the proteins is indicated. E, anti-PP4R4 or preimmune serum was used to immunoprecipitate (IP) endogenous PP4R4 from mouse brain lysate; the immune complexes were separated via SDS-PAGE, transferred to nitrocellulose, and subjected to immunoblotting with the PP4R4 antiserum or with an antiserum reactive to PP4c (Bethyl A300–835A). The position of PP4c is indicated.