Extended Data Fig. 2: Wild-type and truncated DOK7 are detected with similar efficiency by antibodies against the PH and PTB domains in DOK7.
From: Mechanism of disease and therapeutic rescue of Dok7 congenital myasthenia
a, HEK 293 cells were transiently transfected with a plasmid expressing either HA-tagged DOK7 or HA-tagged truncated DOK7 encoded by Dok71124_1127 TGCC dup. Proteins in cell lysates (triplicates) were separated by SDS–PAGE, and western blots were probed with either a rabbit antibody against the PTB domain in DOK7 or a monoclonal antibody against HA (left). We measured the grey levels of the bands for wild-type and truncated DOK7 proteins and normalized the level detected by western blotting with the rabbit antibody against DOK7 with the level detected by western blotting with the antibody against HA. The ratio for wild-type DOK7 was equivalent to the ratio for truncated DOK7 (left), indicating that the rabbit antibody against DOK7 detected wild-type and truncated DOK7 proteins with similar efficiency by western blotting. b, Wild-type and truncated DOK7 were immunoprecipitated with similar efficiency by a goat antibody against the PTB domain in DOK7. HEK 293 cells were transiently co-transfected with plasmids expressing HA-tagged DOK7 and HA-tagged truncated DOK7 encoded by Dok71124_1127 TGCC dup. DOK7 proteins were immunoprecipitated from cell lysates (triplicates) with either a monoclonal antibody against HA or a goat antibody against the PTB domain in DOK7, and western blots were probed with the monoclonal antibody against HA (left). We measured the grey levels, subtracted the level for the background band in the control, non-transfected samples, and normalized the value for each protein immunoprecipitated with the goat-antibody against DOK7 to the value for the same protein immunoprecipitated with the antibody against HA. This ratio was equivalent for wild-type and truncated DOK7 proteins, indicating that the goat antibody against DOK7 immunoprecipitated wild-type and truncated proteins with similar efficiency (right). Plots show individual values from three and four experiments for a and b, respectively, and the mean ± s.e.m. (n.s., not significant); two-sided Student’s t-test.
