Figure 4: TAT-TM2 reduced Aβ levels in vitro and in vivo.

(a) ELISA examination of Aβ levels in the medium of HEK293APP cells transfected with TRPC6 or its mutants within second transmembrane domain (TM2) for 2 days (n=3–6). (b) Lysates of HEK293APP stable cells transfected with C6-mut1 for 2 days precipitated with APP antibody and immunoblotted with indicated antibodies. (c) The Aβ levels in the medium of HEK293APP cells treated with 2 μM indicated peptides for 12 h (n=3–6). (d) Lysates of HEK293APP stable cells incubated with 5 μM TAT-biotin or TAT-TM2-bitoin for 6 h precipitated with avidin beads and immunoblotted with indicated antibodies. (e) ELISA examination in a blinded manner of Aβ levels in the brain lysates of 5 month male APP/PS1 mice intra-peritoneally injected with 200 μl 2.5 mM TAT or TAT-TM2 for 3 h (n=8 mice). (f) The Aβ levels generated in the in vitro purified C99 cleavage assay treated with 2 μM of indicated peptides for 2 h (n=4). (g) Aβ levels in the medium of COS7C99 stable cells treated with 5 μM TAT-TM2 for 12 h (n=3–4). (h) Immunoblots of AICD in HEK293C99 cells treated with 5 μM TAT-TM2 for 12 h or NICD in HEK293APP cells transfected with Notch≜E-myc for 1 day and then treated with 5 μM TAT-TM2 for 12 h. (i) Quantification of the ratio of AICD/C99 or NICD/NotchΔE (n=3–4). CTRL, transfection with YFP. Data were presented as means±s.e.m. of indicated numbers of independent experiments. Two-tailed Student’s t-test was performed for two groups, and one way ANOVA with Newman–Keuls post hoc test was performed for more than two groups. *P<0.05, ***P<0.001 versus CTRL or TAT.