TMEM251 GFP fusion constructs
The plasmid pACGFP-N1 (Clontech, California, USA) was used for obtaining TMEM251 fused to GFP at its C-terminal. TMEM251 cDNA cloned in pTZ57R/T vector was amplified using gene specific primers with flanking recognition sequences for restriction enzymes BamH1 andEcoR1 (Table S4). Ligation of restriction digested PCR amplified TMEM251 cDNA and similarly cut vector was completed using T4 DNA ligase. The recombinant plasmid was transformed into competent E. coli . The bacteria were plated on LB agar containing 100µg/ml of kanamycin and grown at 37°C overnight. Colonies were picked and plasmids were isolated using Qiagen minikit (Qiagen, Hilden, Germany). Insertion of cDNA into vector was confirmed by sequencing. Site directed Ligase Independent Mutagenesis was carried out to introduce the c.133C>T p.(Arg45Trp) variant into the wild-type TMEM251 cDNA cloned in pACGFP-N1 as described.(Chiu, March, Lee, & Tillett, 2004) Briefly, an inverse PCR was done in two reactions to amplify the template plasmid containing the cloned wild-type TMEM251 open reading frame using two tailed long and two short primers (Table S4).