Next, the functional consequences of NBAS mutations on the protein level were investigated. In eukaryotic cells, soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) tethering factors mediate the docking and fusion of transport vesicles with target membranes. Fusion of membranes is mediated by membrane-bound proteins on the transport vesicles (v-SNARE) and target membrane (t-SNARE). NBAS is thought to function as a component of an ER tethering complex that interacts with the t-SNAREs p31, BNIP1, and STX18 at the ER and the v-SNARE Sec22b. This tethering complex also includes ZW10, RINT1, and Sly1 (see Figure S1).9,10 Mutant and control fibroblasts were cultivated in D-MEM media supplemented with 10% FBS, 1% penicillin-streptomycin, and 200 μM uridine at 37°C and 5% CO2. For immunoblots, cells were collected, washed in PBS, and resolved in RIPA buffer.11 10 μg of protein of every sample were separated on a 4%–12% acrylamide gradient gel (LONZA). Primary antibodies (all Sigma-Aldrich) against NBAS (1:2,000), p31 (USE-1) (1:250), and β-actin (1:15,000) were incubated overnight. Enhanced chemiluminescence of proteins was detected with a Vilberscan Fusion FX7. Protein levels were quantified with the software Bio-1D. Testing of individual F1:II.1 fibroblasts showed a significant decrease in NBAS steady-state levels (p = 0.03; two-tailed unpaired t test). Extending the quantification of NBAS protein to affected individuals from families 2–5 and 7 showed a reduction of NBAS levels to 18%–36% in comparison to controls, indicating a substantial impairment of protein translation and/or stability in all seven affected individuals investigated (Figure 3 shows representative findings for individuals from families 1–5). Moreover, the reduction of NBAS was concomitant with a reduction of its proposed interaction partner p31, supporting an important function of NBAS within the syntaxin 18 complex (Figure S1). None of the additional components have been associated with a human disorder. Mouse models of deleted p31 and RINT1 were shown to be embryonic lethal.12,13