After incubation for 60 min at 37 °C, followed by five rounds of washing with PBST, the chromogenic reaction and the following steps were as described above.
After incubation for 60 min at 37 °C, followed by five rounds of washing with PBST, 100 µl aliquots of Hsb-Cap∆41 diluted 1/10, 1/100 or 1/1000 were then added.
After a further incubation of 60 min at 37 °C, followed by five rounds of washing with PBST, 50 µl/well of 3,3′,5,5′-tetramethylethylenediamine 3,3′,5,5′-tetramethylethylenediaminewell Peroxidasolutionrate, KPL) waSureBlue Reserve TMB Microwell Peroxidase Substrate 37 °C.
Briefly, we enriched for N2a cells lacking endogenous PrPC expression (NΔ cells) by five rounds of fluorescence activated cell sorting (FACS) and clonal expansion.
In the first round of selection, phage from the randomized libraries were selected with immobilized LRP6 E3-E4-His protein, followed by five rounds of solution-phase sorting in which the concentration of E3-E4-His was gradually reduced from 300 nM to 0.5 nM, and a 100-fold excess of E3-4-Fc protein was added to deplete antibodies with faster dissociation rates.
The identity of uPAR and β1 integrin interaction was verified further in mock-transfected HCT116 cells by immunodepleting the cell lysates of β1 integrin or uPAR by five rounds of immunodepletion steps.
