Western blot analysis of CaD phosphorylation in the transfected A7r5 cells. The total cell extracts from A7r5 cells transfected with various constructs were immunoblotted with polyclonal anti-pSer527 (Left Panel) and anti-CaD (Right Panel) antibodies. M: Molecular weight markers; samples (Lanes 2-6) are, respectively, cells transfected with A1A2, A3A4, A1234, wtCaD, and EGFP alone (Control). The corresponding ratios of the digitized intensity of the EGFP-tagged CaD variant (the upper red band; Right Panel) to that of the endogenous CaD (the lower red band; Right Panel) are, respectively, 0.93, 1.71, 2.60, 0.43 and, 0 (EGFP); and the corresponding ratios of the digitized intensity of the phospho-EGFP-CaD variant (the upper red band; Left Panel) to that of the phosphorylated endogenous CaD (the lower red band; Left Panel) are, respectively, 0.13 (A1A2), 0, 0, 0.10 (wtCaD) and, 0. The apparent lower signal in the phosphorylation for the engineered variants (human) than the endogenous CaD (rat) may be partly due to different immuno-reactivities of the antibodies. Moreover, since the transfection efficiency is ~35% in all cases, the actual ratios could be higher.