IDP-induced Ca²⁺ oscillations in Jurkat T cells and RS11846 B cells. (A) Single-cell Ca²⁺ recordings showing representative examples of Ca²⁺ oscillations induced by 10μM TAT-IDP or TAT-IDP_DD/AA in more than 50% of Jurkat cells (arrow, peptide addition). (B) Enhancement of TAT-IDP–induced Ca²⁺ elevation by treating Jurkat cells with 2μM pepstatin A for 45 minutes before peptide addition: (i) percentage of cells with Ca²⁺ elevation in response to 10μM TAT-IDP in the presence of DMSO or pepstatin A (mean ± SE, 3 experiments, average 78 cells per treatment per experiment); (ii) amplitude of Ca²⁺ spikes induced by 10μM TAT-IDP (mean ± SE, amplitude of 130 spikes observed in a total of 83 DMSO-treated cells and 299 spikes in a total of 73 pepstatin A-treated cells). (C) Peptide sequences showing the predicted aspartyl protease cleavage site in TAT-IDP and its elimination by the DD/AA substitution in TAT-IDP_DD/AA. (D) Percentage of Jurkat cells displaying each of 3 Ca²⁺ response patterns when untreated or when treated with 10μM TAT-Scr, TAT-IDP, or TAT-IDP_DD/AA (mean ± SE, 6 experiments, average of 80 cells analyzed by 90 minutes of single-cell digital imaging per peptide treatment per experiment). (E) Amplitude of Ca²⁺ spikes during 90 minutes of recordings after 10μM peptide addition to Jurkat cells (mean ± SE, 3 experiments, average 85 cells per peptide addition per experiment). (F) Average Ca²⁺ oscillatory frequency in untreated and peptide-treated Jurkat cells based on analysis of 90 minutes of recordings (mean ± SE, 3 experiments, average 85 cells per peptide treatment per experiment). (G) Single-cell Ca²⁺ recordings showing representative examples of Ca²⁺ oscillations induced by 10μM TAT-IDP or TAT-IDP_DD/AA in more than 50% of RS11846 cells (arrow, peptide addition). (H) Amplitude of individual Ca²⁺ peaks observed in untreated cells and cells treated with 10μM of each peptide (mean ± SE; no spikes in untreated; 34 spikes with TAT-Scr; 104 spikes with TAT-IDP; 248 spikes with TAT-IDP_DD/AA). (I) Frequency of Ca²⁺ oscillations induced by 10μM of each peptide in same dataset analyzed in panel H.