Mutant and control fibroblast cell lines were cultivated at 37°C. 10 μg protein of collected cells were separated on a 4%–12% acrylamide gradient gel, transferred to a PVDF membrane, and immunodecorated with antibodies against NBAS, p31, and β-actin. The antibody for NBAS was detecting a single protein band at approximately 270 kDa corresponding to the predicted molecular weight of NBAS. β-actin was used as a loading control. The quantified protein levels are based on three independent experiments and expressed as percentages of a control cell line, corrected for β-actin. Five of the eight investigated subject cell lines are shown. NBAS and p31 protein levels were severely reduced in fibroblast cell lines of RALF-affected individuals to 21% and 33% of control subjects, respectively. Error bar indicates 1 SD. Protein amount in control 1 was set as 100%.