Fig.3.

Co-IP analysis of DYRK1A-cytoskeleton complexes in lymphoblastoid cell lines (LCLs). A) The levels of DYRK1A and β-actin in crude lysates. RIPA lysates prepared from each test sample were probed for DYRK1A and β-actin by immunoblotting as described in the Materials and Methods. B) Analysis of β-actin co-IP with DYRK1A. RIPA lysates were IP with anti-DYRK1A antibody (R420) and the precipitates were then probed for DYRK1A and β-actin. Mouse anti-rabbit IgG antibody was included with anti-β-actin antibody in immunoblotting as the internal loading control for quantifying precipitates. IgG marks the band of rabbit IgG from antibody R420. C) Analysis of β-actin co-IP with DYRK1A in an individual with asymptomatic carrier PS1 mutation and her normal cousin. Experiments were performed similarly as described in (B). D) Reciprocal analysis of DYRK1A co-IP with β-actin. IP was conducted with anti-β actin antibody with lysates prepared from test samples and then analyzed for DYRK1A similarly as described above in (B). E) The levels of DYRK1A and β-actin in pooled lysates. Each pool was prepared from four distinct cases and analyzed as described in (A). The relative ratio of DYRK1A and β-actin in DS and AD to that of the control was plotted. Control (100%) was indicated by a dotted line. Difference to the control with statistical significance at the level of p < 0.05 was marked with asterisk. F) Quantification of β-actin co-IP with DYRK1A in pooled lysates. Co-IP experiments were conducted with pooled samples similarly as described in (B). The relative ratio of DYRK1A and β-actin in DS and AD was quantified similarly as described in (E). G) Quantification of β-actin and α-tubulin IP and co-IP in pooled samples. Pooled samples were first IP using either anti-β actin or anti-α-tubulin antibody and the precipitates were then analyzed for IP and co-IP with the congruent and the counter rabbit polyclonal antibody, respectively. The plots shown are the ratio of actin and tubulin IP and co-IP in AD relative to that of the control. H) Quantification of β-actin co-IP with α-tubulin in pooled lysates. Pooled samples were first IP using anti-α-tubulin and then analyzed for α-tubulin and β-actin as described in (F). The relative ratio of β-actin and α-tubulin in DS and AD was presented.