Figure 4
From: Scribble co-operatively binds multiple α1D-adrenergic receptor C-terminal PDZ ligands
SNAP-α1D-adrenergic receptor C-terminal PDZ ligand:GST-SCRIB pulldown assays indicate a co-operative binding model. (A) PAGE NIR of HEK293 cell lysates transfected with vehicle alone (− vector), pSNAP vector (SNAP), N-terminal SNAP-tagged α1D-AR C-terminus (α1D-CT), α1A, α1B and α1D-AR. (B) Coomassie stain of GST-SNAP-α1D-CT purification ± IPTG induction, unbound (FT), bound to beads (B), following TEV cleavage (+TEV) and anion exchange chromatography (AEX). (C) PAGE NIR of purified SNAP-α1D-CT pre-labeled with BG-782. (D) SNAP-α1D-CT standard curve plotting concentration of BG-782 labeled SNAP-α1D-CT versus fluorescence quantified at λ = 800 nm. (E–G) Representative PAGE NIR gels of SNAP-α1D-CT pulldowns with GST-SCRIB (E), GST-PDZ4 (F), or GST-PDZ1 (G). (H) Concentration-response curves quantifying SNAP-α1D-CT bound to GST-SCRIB, SCRIB truncated before PDZ domain (ΔPDZ4), before PDZ domain 3 (ΔPDZ34), SCRIB PDZ1, or SCRIB PDZ4 (mean ± SEM, n = 3–4). ***p < 0.001, One-way ANOVA with Tukey’s post-hoc test.
