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. 2015 Oct 22;6(10):e1939. doi: 10.1038/cddis.2015.307

Figure 4.

Figure 4

Design of a CPP able to specifically inhibit Kidins220 calpain processing induced by excitotoxicity. (a) Cell-membrane peptide Tat-K contains from the N- to the C-terminus amino acids 47–57 of the HIV-1 Tat protein (italic) followed by two proline linker residues and rat Kidins220 amino acids 1668–1681 (bold), which include the major calpain cleavage site (arrow). Control peptide Tat-S is similar to Tat-K but Kidins220 amino acids (bold) are randomly organized. (b) Analysis of Tat-K interference of in vitro calpain processing of Kidins220. Protein extracts from cortical neurons were subjected to in vitro digestion with purified calpain I (20 U/ml) for 15 min in the presence of peptides Tat-K (K) or Tat-S (S) (0.5 μM). Results were analyzed by immunoblot with Kidins220 (Kid-Ct), spectrin and NSE antibodies. (c) Establishment of optimum conditions for peptide entry into cortical neurons. Primary cultures of neurons (DIV 14) were incubated with 5, 10 or 25 μM of FITC-labeled control peptide (FITC-Tat-S) for 2, 4 or 6 h and pictures were taken in a conventional fluorescence microscope. Scale bar, 25 μm. (d) Dose response of Tat-K-specific interference of Kidins220 calpain processing induced by NMDAR overactivation. Cortical neurons were pre-incubated for 1 h with 10 or 25 μM of Tat-K (K) or Tat-S (S) before NMDA addition for 4 h and analyzed by immunoblot with Kidins220 (Kid-Ct), spectrin and NSE antibodies. (e) Time course of Tat-K-specific interference of Kidins220 calpain processing induced by excitotoxicity. Neurons were pre-incubated for 1 h with 25 μM of Tat-K (K) or Tat-S (S) before NMDA addition for 2, 4 or 6 h and analyzed as before. (f) Quantitation of Tat-K protection of Kidins220 processing induced by excitotoxicity. Levels of Kidins220 were established by densitometric analysis of immunoblots using NIH Image software and normalized to those of NSE present in the same samples. For each time point, Kidins220 levels in neurons treated with NMDA in the presence of Tat-S or Tat-K are represented as relative values to those obtained in cultures incubated with the same peptide but no NMDA, arbitrarily given a 100% value. Average of five independent experiments with S.E.M. is given. Statistical significance was determined by paired Student's t-test (**P<0.01, ***P<0.001)