Protein expression
HEK-293T cells (4 x 105) were cultured in Dulbecco’s
Modified Eagle Medium (DMEM, GIBCO) supplemented with 10% fetal calf
serum (FCS, GIBCO). Cells were transfected with plasmids encoding wild
type (WT) or mutant ACT1 (p.D451G, p.K454fs11*, or p.T536I already
reported [12] serving as positive control), using Lipofectamine 2000
according to the manufacturer’s protocol. After 24 hours, cells were
lysed with NP40 lysis buffer (1M Tris-HCl, 5M NaCl, 1% NP40). Samples
were separated by SDS-PAGE on a 4-20% gradient gel (BioRad, 456-1093),
and proteins were transferred to a PVDF membrane (Millipore). The
membrane was blocked using 5% bovine serum albumin with 0.1% Tween 20
in PBS, followed by incubation with the primary antibodies against ACT1
and GAPDH. Visualization of the proteins was assessed by enhanced
chemiluminescence.