Tau sites phosphorylated by GSK-3β in an in vitro reaction. Thirteen phosphorylation-site-specific antibodies were used to probe dot blots of GSK-3β phosphorylated tau. Panel A and B show two representative blots. Three repetitions of each phosphorylated tau concentration, ranging from 800 to 1.56 ng, were spotted on a blot, then probed with an anti-phosphorylation specific antibody (See Materials and Methods for details). To estimate non-specific background levels, each blot also included one spot of non-phosphorylated tau at each concentration. Following density analysis, the antibody titrations were plotted (Panels D and E) and 1/2 max concentrations were estimated. Anti-S422 antibody shown in Panel C, and its titration plot in Panel F, did not recognize phosphorylation and served as a negative control.