Ecl prime chemiluminescent reagent

Whole-cell lysates were prepared in IP Lysis Buffer (Pierce) containing
protease and phosphatase inhibitors. Proteins were separated on 10%
SDS-polyacrylamide gels and transferred to PVDF. Membranes were blocked with 5%
non-fat dry milk in TBS-T and subsequently probed with 1:1000-fold dilution of
anti-PSAT1 (Proteintech, 10501–1-AP) or anti-PHGDH (Sigma HPA021241)
antibodies for 16 hours at 4°. Washed membranes were then incubated with
1:5000 dilution of HRP-conjugated anti-rabbit or anti-mouse secondary
antibodies. Protein detection was done by exposure to ECL Prime chemiluminescent
reagent (GE Healthcare). Protein loading was assessed using anti-β-actin
antibody (Sigma, A2228). Densitometry was performed via ImageJ.

The 37 amino acid cytoplasmic domain peptide of integrin β1 was coupled to CNBR-activated sepharose beads (GE Biosciences, Pharmacia). Beads were incubated overnight at 4 °C with 5 µg of pFBLN1 or recombinant fibulin-1C (rFBLN1C), purified from conditioned media of transfected HT1080 cells ^{28 (link)}, in TBS binding buffer containing 25 mM OG and 1mM of CaCl_² and MgCl_² . The supernatant (unbound fraction) was removed and beads were washed 4 times with binding buffer and 2X non-reducing sample buffer was added. Bound and unbound fractions were resolved on 4–12% NUPAGE gel, transferred to PVDF membrane and probed with anti fibulin-1D’ antibody (Rb1603). Visualization was performed using ECL prime chemiluminescent reagent (GE Healthcare Life Sciences). Re-probing with the 5D12 antibody was done after washing the membrane with 50 mM glycine pH 2.3 for 10 minutes to remove bound antibodies then rinsing with copious amounts of TBS buffer containing 0.1% Tween 20 (TBST).