Fig. 2

(A) Expression of canonical CD59 and (B) its isoforms: IRIS-1, and IRIS-2 in human neuroblastoma cells line (SH-SY5Y) undifferentiated and differentiated into mature neurons with BDNF and retinoic acid analyzed by semi-quantitative RT-PCR (n = 3). The negative control is the reaction mix, without template (no cDNA). (C) GAPDH served as a control. (D) Western blot analysis of IRIS-1 protein level in undifferentiated vs. differentiated SH-SY5Y cells, showing increased expression post-differentiation (n = 6). β tubulin was used as a loading control. The quantification is shown in (E). (F) Similarly, IRIS-2 protein levels were higher in differentiated cells, as shown by Western blotting (n = 6). Quantification is shown in (G). (H) CD59 expression in SH-SY5Y cells was measured by QRT-PCR to verify the mRNA level of CD59 knockdown (n = 3). Non-targeting negative control siRNA (Ambion, #4390843) was used as a negative control. GAPDH was used as a reference gene. (I) Western blot for CD59 protein to confirm knockdown efficiency for repeats from both the noradrenaline secretion assay- and p-tau/Cdk5 expression experiments, with β-tubulin as a loading control (n = 11). Quantification shown in (J). (K, M) Western blotting assessed IRIS-1 and IRIS-2 protein levels post-knockdown, respectively (n = 11, each). Quantifications are shown in (L and N). Statistics (in E, G, H, J, L, and N): two-tailed student T-test. Error bars indicate SD