Supplementary Figure 7 Phosphorylation of SH2 peptide AtzA fusion (pY-AtzA) by Src kinase

Article: Stimulus-responsive Self-Assembly of Protein-Based Fractals by Computational Design Pre-print: bioRxiv 274183; doi: https://doi.org/10.1101/274183 Figure: S7. Phosphorylation of SH2 peptide AtzA fusion (pY-AtzA) by Src kinase. In order to verify phosphorylation of AtzA by Src kinase into phosphorylated SH2 peptide AtzA fusion (pY-AtzA), ELISA with (1:4000 dilution) antiphosphotyrosine-horseradish peroxidase conjugate was performed on pY-AtzA samples either with Src kinase (+) or without Src kinase (-), in phosphorylation reaction buffer at 1.25 µg/mL pY-AtzA or 20 µg/mL pY-AtzA. Data is presented as mean ± 1 standard deviation. (SI 2.9) Phosphorylation, assembly formation, and disassembly – The phosphorylation protocol was based upon Src kinase activity assay by Sigma (Catalog # S1076). In a final reaction volume of 150μL, 3μM AtzAM1 was mixed into 1X Kinase Activity Buffer (4mM MgCl2, 2.5mM MnCl2, 0.25mM DTT, 5mM MOPS, 2.5mM glycerol-2-phosphate, 1mM EGTA, 400nM EDTA, pH 7.6), 2.5 mM MnCl2,HNG, 2 mM ATP, 800ng Src kinase, and incubated for 7 – 16 hr at 25°C for phosphorylation to occur. After phosphorylating, AtzCM1 was added to a final 2μM concentration. Assembly was allowed to form at 2hr 25°C. Disassembly was performed by adding 4.8μg of YopH phosphatase into the 150μL reaction mixture after assembly formation occurred. Size measurements using DLS were performed to determine assembly formation/disassembly. (SI 2.7) Enzyme-linked immunosorbent assay (ELISA) – Phosphorylated AtzAM1 (pY27 AtzAM1) was loaded onto clear flat-bottom immuno 96-well plates (Thermo Scientific item #442404) at 20μg/mL and 1.25μg/mL in 50μL 1X PBS (Gibco pH 7.4, #10010023) overnight at 4◦C. Plates were rinsed twice in 200μL 1X TBS (Biorad #1706435). 1% BSA in TBS 0.05% Tween 20 was used to block wells at 200μL block solution for 1.5hr at 25°C under gentle agitation. Anti-phosphotyrosine 4G10 Platinum HRP conjugate (EMD #16-316) was diluted 1:5000 in 1% BSA TBS 0.05% Tween 20 and loaded onto the well at 25°C for 1.5hr under gentle agitation. Excess anti-phosphotyrosine was washed off with 200μL of TBS 0.05% Tween 20 in triplicate. To detect bound antibody, 100μL of TMB substrate reagent (Biolegend #421101) was added to each well and incubated for 5 minutes at 25°C. 100μL of TMB stop solution (Biolegend #423001) was added to the wells. Absorbance was read at 450nm using the Tecan Infinite M200 Pro plate reader.****Note: This work was part of the <b>Rutgers Biomod 2016 </b>project (M. Liu, A. Permaul, O. Dineen, M. Khalid, M. Shea, G.L. Bilker). See reference for additional details.