Co-Immunoprecipitation
HEK-293T cells (1 x 106) were co-transfected with an
N-terminal HA-tagged plasmid encoding WT or mutant (p.D451G, p.K454fs11*
or p.T536I) ACT1 along with a C-terminal Flag-tagged plasmid encoding
IL-17RA. After 24 hours, cells were lysed as above. An aliquot of the
lysate was reserved for the input control and the co-immunoprecipitation
was performed on the remaining lysate using agarose beads with anti-Flag
antibodies conjugated (Sigma-Aldrich, F2426) followed by a 3-hour
incubation and washing with NP40 lysis buffer. Western blots were
performed as above using primary antibodies against HA (R&D systems,
HAM0601) or Flag (Sigma-Aldrich, A8592).