Recombinant protein cell binding assay
100 nM recombinant MBP control, MBP tagged-CCP21 or MBP tagged-CCP22 was
coated onto a 96 well tissue culture plate. Non-specific binding was
blocked using DMEM containing 10 mg/mL bovine serum albumin, 10 mM
HEPES. 20,000 HEK293 control (α4/α9-), integrin α4β1
(α4β1+), or α9β1 overexpressing
(α9β1+) cells were seeded onto the coated plates in
blocking buffer. The α4β1+ cells were incubated for 3
hours and the α9β1+ cells were incubated for 30
minutes at 37⁰C. and incubated at 37⁰C for 30 minutes. Plates were
washed, fixed with 4% PFA and visualised using DAPI. The number of
adhered cells was measured using Fiji.