Extended Data Fig. 1: Concentration dependence of various cationic and non-cationic peptides on SEP-GluA1 fluorescence.

(a) Sample flow cytometry image indicating side scatter vs. forward scatter, and selection of gates to isolate cells. (b) Distribution of cells by FITC signal, normalized to the maximum number of cells found within a given FITC range bin. ZIP shifts the distribution of this curve to the left. (c) Effect of heparin on the ZIP-induced reduction in SEP-GluA1 fluorescence, added either to the same tube as ZIP before adding to cells, or added to the cell culture independently before ZIP. Heparin completely blocked ZIP or TAT’s effects on SEP-GluA1 fluorescence, whether heparin was premixed with peptide – vehicle vs. heparin/ZIP p = 0.39; vehicle vs. heparin/TAT p = 0.93 – or first added to the cells – vehicle vs. heparin/ZIP p = 0.17; vehicle vs. heparin/TAT p = 0.79. (d) Ratio of SEP-GluA1 internalization as a function of the log concentration of scrZIP and myr-scrZIP. scrZIP all n = 3, myr-scrZIP all n = 3. (e) Effect of 100 μM V5 or FLAG peptides relative to 100 μM ZIP on SEP-GluA1 fluorescence. Vehicle vs. V5 p = 0.87; vehicle vs. FLAG p < 0.0001. (f) Concentration dependence of the AIP peptide on SEP-GluA1 fluorescence. Vehicle vs. 10 μM AIP p < 0.0001; vehicle vs. 100 μM AIP p < 0.0001; vehicle vs. 1 mM AIP p < 0.0001. (g) Concentration dependence of the scrZIP peptide on SEP-GluA1 fluorescence. Vehicle vs. 0.1 μM scrZIP p = 0.32; vehicle vs. 1 μM scrZIP p < 0.0001; vehicle vs. 10 μM scrZIP p < 0.0001; vehicle vs. 100 μM scrZIP p < 0.0001; vehicle vs. 1 mM scrZIP p < 0.0001. (h) Concentration dependence of the Arg9 peptide on SEP-GluA1 fluorescence. Vehicle vs. 10 μM Arg9 p < 0.0001; vehicle vs. 100 μM Arg9 p < 0.0001. (i) Concentration dependence of the non-cationic AA3H and AA3H-PLP peptides on SEP-GluA1 fluorescence. Vehicle vs. 10 μM AA3H p < 0.0001; vehicle vs. 100 μM AA3H p < 0.0001; vehicle vs. 10 μM AA3H-PLP p < 0.0001; vehicle vs. 100 μM AA3H-PLP p < 0.0001. (j) Relationship between the net charge of each peptide and the relative change in SEP-GluA1 fluorescence (at 100 μM peptide), r² = 0.70, p < 0.0001. Error bars are centered at the mean, ± 1 s.e.m., and full statistics are provided in Supplemental Table 1.