14-3-3 binding inhibits αTAT1 association with Importin-α. (a–d) (a) Normalized corecruitment levels by indicated baits of mCherry (α-TAT1: 79, T322A: 45, ST/A: 49), mCherry-14-3-3β (α-TAT1: 64, T322A: 63, ST/A: 66), mCherry-14-3-3γ (α-TAT1: 50, T322A: 57, ST/A: 64), mCherry-14-3-3ε (α-TAT1: 65, T322A: 52, ST/A: 58), mCherry-14-3-3ζ (α-TAT1: 70, T322A: 58, ST/A: 79), mCherry-14-3-3η (α-TAT1: 48, T322A: 50, ST/A: 58), mCherry-14-3-3θ (α-TAT1: 53, T322A: 68, ST/A: 53), mCherry-14-3-3σ (α-TAT1: 53, T322A: 56, ST/A: 73); (b) normalized recruitment levels of baits (α-TAT1: 90, T322A: 97, ST/A: 105); (c) kinetics of mVenus-FKBP-α-TAT1 and mCherry-14-3-3-ζ translocation to plasma membrane; n = 24 cells, means ± 95% confidence interval are shown; (d) normalized corecruitment levels in cells coexpressing myc-miRFP or myc-miRFP-Difopein by mVenus-FKBP-α-TAT1 of indicated preys mCherry-KPNA2 (miRFP: 27, Difopein:39), mCherry-KPNA4 (miRFP: 25, Difopein:37), mCherry-KPNA6 (miRFP: 26, Difopein:38). ***, P < 0.001 or as shown, Student’s t test.