Endogenous SET translocation after 5 h 30 min of Jcasp peptide internalization in mouse primary neurons. A) Immunocytochemical staining (epifluorescence) of endogenous SET (Cy3) in primary neurons treated with the Jcasp peptide (2 μM) for 5 h 30 min, and control neurons in the absence of any peptide. Nuclei were labeled with DAPI. SET is mostly present in the nucleus of control neurons in the absence of any peptide as indicated in combined images (Merge), whereas SET translocation to the cytoplasm occurs after the internalization of Jcasp peptide (see arrowheads). One representative immunocytochemical staining out of five independent experiments is presented. Scale bar, 10 μm. B) Representative western blot of cytoplasmic (Cyt) and nuclear (N) neuronal extracts obtained by sub-cellular fractionation (n = 3 independent experiments). Endogenous SET was detected with a SET antibody. Dab1 was used as a cytoplasmic control and histone was used as a nuclear control. A total of 10 μg of protein was loaded in each lane. Endogenous SET at 39 kDa is partially localized in the cytoplasm in cells containing Jcasp peptide. Cleaved SET fragments cannot be detected. C) Relative expression of SET mRNA assessed by quantitative real-time PCR in cells treated with Jcasp peptide for 1 h 30 min, 2 h 30 min, 3 h 30 min and 5 h 30 min and with the Penetratin (Pen) peptide only at the same time points. Results are expressed as the ratio of SET mRNA in Jcasp peptide internalization versus Penetratin only assays at the different time points. Data presented are the mean ± SEM of three independent experiments. Jcasp peptide does not affect the level of SET mRNA.