EFEMP1 expression vector
To evaluate the cellular expression and localization of
disease-associated EFEMP1 variants we used a pENTR/D-TOPO entry
vector (Invitrogen, Carlsbad, CA) with cloned wild-type EFEMP1cDNA (provided as a gift from Dr. Rosario Fernandez-Godino, (Garland et
al., 2021). The EFEMP1 mutants (p.Asn80Tyr, p.Arg140Trp,
p.Arg345Trp, p.Arg477Cys) were created using the QuikChange
Site-Directed Mutagenesis kit (Agilent Technologies, Santa Clara, CA).
To create the p.Ter494Glnext*29 mutation, a gBlocks™ Gene Fragment
(Integrated DNA Technologies, Iowa) was designed and cloned in to a
pENTR/D-TOPO entry vector. The wild-type and mutant entry clones were
verified by sequencing analysis and moved into a Gateway destination
expression vector modified to contain an N-terminal V5 epitope tag
in-frame (pCAG-V5-IRES-EGFP) (Zhang et al., 2012) by LR recombination.
Plasmid DNA was purified using the EndoFree Plasmid Maxi kit (Qiagen,
Valencia, CA).