Immunoprecipitation
Constructs expressing ITGA9-GFP or ITGA4 were transfected into HEK293WT
or HEK293 cells overexpressing SVEP1-FLAG. 48 hours post transfection
the transfected cells were scraped into lysis buffer (mmol/L-1: 50
Tris-HCL, 150 NaCl, 1 EDTA, 1% Triton-X-100 and 1x phosphatase and
protease inhibitors). Lysates were incubated on ice (15 minutes),
sonicated, and cleared by centrifugation at 17,000 g . Anti-FLAG
– agarose beads (Sigma Aldrich) were prepared by washing 3x in wash
buffer (mmol/L-1: 50 Tris-HCL, 150 NaCl and 1 EDTA).
Cell lysate was added to the pelleted beads. The IP reactions were
incubated for 90 minutes at 4°C with agitation. The pulled down proteins
were denatured from the beads using 25 µL of a solution containing 50%
4x lauryl dodecyl sulphate sample buffer, 45% wash buffer, 5%
β-mercaptoethanol. The ITGA9-GFP was detected in a western using an
anti-GFP antibody (Thermo Scientific cat# A-11120). The ITGA4 proteins
were detected in a western using an anti-integrin α4 antibody (Santa
Cruz cat# sc-365209).