Journal: Disease Models & Mechanisms

Article Title: A homozygous FITM2 mutation causes a deafness-dystonia syndrome with motor regression and signs of ichthyosis and sensory neuropathy

doi: 10.1242/dmm.026476

Figure Lengend Snippet: Identification of a genetic defect underlying syndromic hearing impairment in family W09-1008 and expression analysis of wild-type and p.Glu2* FITM2 fused to a FLAG-tag in HEK293T cells. (A) Partial sequences of FITM2 exon 1 are shown from an affected member, an unaffected heterozygous sib and an unaffected wild-type sib of family W09-1008. The predicted amino acid changes and the surrounding amino acids are indicated above the sequence. Sequence NM_001080472.1 was employed as a reference. (B) The left panel shows a western blot of a gel on which 10% of the cell lysate was loaded (before affinity purification). The right panel shows a western blot of a gel on which 10 µl of the lysate after affinity purification (anti-FLAG) was loaded. Wild-type (WT) FITM2 migrates around 29 kDa and it is absent upon transfection and expression of the p.Glu2* FITM2 construct. After affinity purification, a very weak band is observed at ∼16 kDa. However, the intensity of the 16 kDa band is about 2700-fold lower than the wild-type FITM2 band and therefore it is likely to have little or no biological impact. The gel was immunostained with an anti-FLAG polyclonal antibody. Of four ATG-triplets in the original reading frame, three (codon positions 94, 493 and 508) are predicted to be potential translation initiation sites by the Netstart 1.0 algorithm ( Pedersen et al . , 1997 ). Accordingly, alternative proteins would consist of 285 amino acids (aa) (26.2 kDa), 152 aa (11.2 kDa), and 147 aa (10.6 kDa), respectively. Expression constructs encode FITM2 fused to a C-terminal Strep-FLAG-tag (SF-TAP), adding approximately 6 kDa to the proteins. Wild-type FITM2 was found to migrate according to molecular weight of ∼29 kDa, which is lower than the predicted mass of the complete protein. However, fragments of similar length are observed with anti-FITM2 staining in the work of and pro-peptide cleavage is predicted by the ProP algorithm. Marker size is indicated between the panels and given in kDa.

Article Snippet: For DNA transfections, HEK293T cells were seeded in 6-well plates, grown overnight, and transfected with 2 μg of plasmid using PEI transfection reagent (Merck Millipore).

Techniques: Expressing, FLAG-tag, Sequencing, Western Blot, Affinity Purification, Transfection, Construct, Molecular Weight, Staining, Marker