Figure 1 of
Tucker, Mol Vis 2004;
Figure 1. Catalytic domain mutations L954P and P858S have dramatic effects on RetGC-1 functioning
P858S and L954P (catalytic domain) substitutions have severely reduced RetGC-1 activities. Membranes from transiently transfected HEK 293 cells were assayed for RetGC-1 activity for 20 min in GC buffer (described in methods). Equivalent total protein amounts of pRC-CMV, RetGC-1 in pRC-CMV, GC-1 P858S, or GC-1 L954P were immunoblotted with an antibody to the KHD of RetGC-1 (inset). For GCAP-stimulated samples, 17 μM GCAP-1 or GCAP-2 and 1 mM EGTA was added. Mn/Tx100 labeled samples contained 1% Triton-X-100 and 10 mM MnCl2 instead of MgCl2. PRC labeled samples represent HEK293 cells transfected with the empty expression vector pRC-CMV.