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).