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. 2019 Jan 3;8:e39180. doi: 10.7554/eLife.39180

Figure 2. PICK1 binding on SCMS is specific and the binding strength is increased two orders of magnitude compared to in-solution affinities.

Schematic and representative confocal images demonstrating saturation with labeled PICK1 (a) and competition between labeled PICK1 and unlabeled PICK1 (b) on SCMS expressing TAC-YFP-DAT. Brackets indicate the varied PICK1 pool. (c) Normalized binding as a function of unlabeled PICK1 concentration (black), left axis (Kd*=47 ± 5 nM) (n = 7) compared to in-solution based FP-measurements (grey), right axis (Kd = 9± 2 μM, n = 3, performed in triplicates). (d) Normalized binding as a function of unlabeled PICK1 concentration (black), left axis (IC50 = 29 ± 4 nM) (n = 5) compared to in-solution based FP-measurements (grey), right axis (Ki,app = 2 ± 0.4 μM, n = 3, performed in triplicates). Scale bars 10 μm.

Figure 2.

Figure 2—figure supplement 1. The PICK1 – TAC-YFP-DAT interaction is saturable and depends primarily on the PDZ binding motif.

Figure 2—figure supplement 1.

(a–b) Representative images demonstrating PICK1 time series (or end points) on SCMS expressing the TAC-YFP-DAT or TAC-YFP-DAT+A constructs. (c) Normalized PICK1 binding as a function of incubation time. For TAC-YFP-DAT half maximum binding is 19 ± 3 min, (n = 3).
Figure 2—figure supplement 2. PSD-95 PDZ1-2 binding to SF-β1AR in supported cell membrane sheet.

Figure 2—figure supplement 2.

(a) Concentration dependent normalized binding of PSD-95 PDZ1-2 (black) to β1AR (Kd*=3 ± 2), n = 3. (b) Representative images demonstrating a PSD95-PDZ1-2 concentration series on supported cell membranes expressing the β1-AR.
Figure 2—figure supplement 3. The intrinsic PDZ affinity does not translate directly to avidity but determines maximal binding.

Figure 2—figure supplement 3.

(a) Quantification of concentration dependent binding of PICK1 to TAC-YFP-GluA2 and TAC-YFP-DAT, plotted on a linear scale. TAC-YFP-DAT Bmax = 100%, TAC-YFP-GluA2 Bmax = 44 ± 9%, (****p<0.0001; n = 3 and 7, respectively). (b) Representative images of end points of the PICK1 concentration series to TAC-YFP-DAT and TAC-YFP-GluA2.