Purified proteins were detected using a polyclonal RALA Antibody (#3526S, Cell Signaling Technology, Danvers, MA, USA) at a dilution of 1:1000, and an anti-rabbit IgG secondary antibody (#926–32211, IRDye 800CW Goat anti-Rabbit IgG, Li-cor, Lincoln, NB, USA) at a dilution of 1:20,000. Proteins were also detected using a 6x-His Tag monoclonal antibody (#MA1-21315, ThermoFisher Scientific, Waltham, MA, USA) at a dilution of 1:1000, and an anti-mouse IgG secondary antibody (#102673–408, VWR, Radnor, PA, USA) at a dilution of 1:20,000. These antibodies were used to confirm protein levels and determine appropriate binding of the RALA Antibody (See panel B of S9 Fig ). An Odyssey CLx Imaging System (Li-cor, Lincoln, NB, USA) was used to visualize the Western. Relative quantification of the image was performed using Image J (https://imagej.net/ ).
We note that while we attempted to study the effects of all variation observed here, Proband 10 was identified after functional validation began, and the recombinant protein with the K128R variant (observed in probands 6 and 7) was not able to be expressed and purified consistently. Thus GTPase and G-LISA experiments were not performed using K128R or A158del mutants.
We note that while we attempted to study the effects of all variation observed here, Proband 10 was identified after functional validation began, and the recombinant protein with the K128R variant (observed in probands 6 and 7) was not able to be expressed and purified consistently. Thus GTPase and G-LISA experiments were not performed using K128R or A158del mutants.