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. 2019 Dec 20;15(2):1706026. doi: 10.1080/15592324.2019.1706026

Figure 2.

Figure 2.

VIP1 interacts with PP2A-C subunits in vitro.

(a) Pull-down assay with full-length VIP1 (VIP1 FL), His-tagged PP2A-A2 (His-PP2A-A2), His-tagged PP2A-C3 (His-PP2A-C3), and Myc-tagged PP2A-C5 (Myc-PP2A-C5). Either GST alone (GST-Alone) or the GST-VIP1 FL fusion protein was bound to the Glutathione Sepharose 4B resin (GE Healthcare, Little Chalfont, UK), reacted with His-PP2A-A2, His-PP2A-C3, Myc-Alone, or Myc-PP2A-C5, and then eluted from the resin. GST-fused proteins, His-tagged proteins and Myc-tagged proteins in the resulting solutions were detected by western blotting with a horseradish peroxidase (HRP)-conjugated GST antibody (Fujifilm Wako Pure Chemical Co., Osaka, Japan), HisProbe-HRP (Thermo Fisher Scientific Inc., Waltham, MA, USA), and anti-Myc-tag pAb (Medical & Biological Laboratories CO., Nagoya, Japan), respectively. Experiments were performed three times and a representative result is presented. Protein masses (kDa) are indicated on the left. (B) Pull-down assay with the N-terminal region (1–186 amino acids) of VIP1 (VIP1 N) and its C-terminal region (165–341 amino acids, VIP1 C). Either GST-fused VIP1 N (GST-VIP1 N) or GST-fused VIP1 C (GST-VIP1 C) was bound to the resin and reacted with either His-PP2A-C3 (left panel) or Myc-PP2A-C5 (right). These proteins were then detected as in the panel A. Experiments were performed three times and a representative result is presented. Protein masses (kDa) are indicated on the left.