Western blots
To quantify transfected EFEMP1 protein, cells were harvested 48
hours post transfection (average transfection efficiency 75%) (Table
S1). Whole cell lysates were prepared by careful aspiration of condition
media and adding cold RIPA buffer (Sigma-Aldrich, St. Louis, MO) and
Pierce Protease Inhibitor (Thermofisher, Waltham, MA) to each well and
agitating for 30 minutes at 4°C. After incubation, cells were
transferred to Eppendorf tubes and centrifuged for 30 minutes at 4°C.
The supernatant was retrieved and the protein concentration was
determined using the Pierce™ BCA Protein Assay Kit (Thermofisher,
Waltham, MA). For each vector group, 5ug of protein was mixed with 4X
Protein Sample Loading Buffer (Li-Cor Biosciences, Nebraska) and water
for a total volume of 12ul. Each sample was denatured by heating at 95
°C for 3 minutes and loaded in to a 15 well 4–20% Mini-PROTEAN® TGX™
Precast Protein Gel (Bio-Rad, Hercules, CA). Electrophoresis was
performed at 120v for approximately 1.5 hours. The gel was retrieved and
transferred to a PVDF blotting membrane using the iBlot™ 2 system
(Invitrogen, Carlsbad, CA). For normalization of protein loading, total
protein staining was done using the Revert™ 700 Total Protein Stain Kit
(Li-Cor Biosciences, Nebraska), following the manufacturer’s protocol.
After imaging in the 700nm channel using the Odyssey® Imaging System
(Li-Cor Biosciences, Nebraska), the membrane was blocked for 1 hour at
room temperature using Intercept® Blocking Buffer (Li-Cor Biosciences,
Nebraska), then overnight incubation with Anti-Fibulin-3 Antibody
(mab3-5): sc-33722 (Santa Cruz Biotechnology, Dallas, TX) diluted with
blocking buffer to a final concentration of 1:100. After overnight
incubation, the membrane was incubated in IRDye® 800CW secondary goat
anti-mouse antibody (Li-Cor Biosciences, Nebraska), at a final
concentration of 1:10,000 for 1 hour and then imaged in the 800nm
channel. Western blot quantification and analysis was carried out using
ImageStudioLite® Software (Li-Cor Biosciences, Nebraska). Relative
EFEMP1 abundance was calculated as the ratio between the EFEMP1 signal
and the corresponding total protein signal for each sample. Three
biological repeats were done for each EFEMP1 condition. Differences
between mutant and wildtype EFEMP1 were tested using Student’s t-test
with Bonferroni correction.