Chitin resin

Wild-type S. flexneri corresponds to the serotype 2a 2457T strain originally isolated in 1954 [27 (link)]. The S. flexneri Spa47 null strain was engineered by Abdelmounaaïm Allaoui as described by Jouihri et al [28 (link)]. Rabbit polyclonal antibodies against IpaC and IpaD and the pWPsf4 expression plasmid were generous gifts from William Picking and Wendy Picking (University of Kansas). E. coli strains and 2x ligation mix were from Novagen (Madison, WI). Restriction enzymes, the pTYB21 protein expression plasmid, polymerase chain reaction (PCR) buffer, Phusion High-Fidelity polymerase, and chitin resin were purchased from New England Biolabs (Ipswich, MA). Oligonucleotide primers and the synthesized spa47 gene were from Integrated DNA Technologies (Coralville, IA). The N-terminal GFP pWPsf4 vector was designed in house and synthesized by General Biosystems (Morrisville, NC). The Superdex 16/600 size exclusion and 5-ml Q FF columns were purchased from General Electric (Pittsburgh, PA). ATP was from Sigma-Aldrich, and α-³²P-ATP was from PerkinElmer Life Sciences. Dithiothreitol (DTT) and ampicillin were from Gold Biotechnology (St. Louis, MO). Defibrinated sheep red blood cells were from Colorado Serum Company (Denver, CO). All other solutions and chemicals were of reagent grade.

C₁₆ derivatives and BODIPY®-TMR labeled derivatives of PtdInsPs (PtdIns[3]P, PtdIns[4,5]P₂, and PtdIns[3,4,5]P₃) were obtained as from Echelon Biosciences (Salt Lake City, UT). 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) and 1,2-dioleoyl-sn-glycero-3-{[N-(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl} nickel salt (DGS-NTA(Ni)) were purchased from Avanti Polar Lipids (Alabaster, Alabama). BL21(DE3) competent Escherichia coli cells were purchased from Invitrogen whereas BL21(DE3) Rosetta competent E. coli cells were from VWR International (Darmstadt, Germany). Chitin resin was obtained from New England Biolabs (Ipswich, MA) and NTA(Ni²⁺) agarose (Protino™) from Macherey-Nagel (Düren, Germany). Antibodies specific for CB2 (1: 1000, Cat. No. 261 011) and NL2 (1: 1000, Cat. No. 129 213) were purchased from Synaptic Systems (Göttingen, Germany) whereas the His-tag antibody (1: 1000, ab18184) was obtained from Abcam (Cambridge, UK). Silicon wafers were purchased from Silicon Materials (Kaufering, Germany). 1,1,1-Trimethyl-N-(TMS)silanamine (HMDS) was purchased from VWR International (Darmstadt, Germany).

Tn5 transposase enzyme was extracted according to Picelli et al., 2014 with a few modifications^{29 (link)}. Briefly, the pTXB1-Tn5 vector (Addgene) was transformed into T7 lysIq competent cells (NEB). Single colonies were picked and two litres of bacterial culture in LB was grown at 37 °C; when the cultures reached OD 0.9, cells were induced with IPTG 0.25 mM and grown at 24 °C until OD 2.5. Cells were collected and resuspended in 100 ml HEGX buffer (10 mM Tris-HCl pH 7.5, 700 mM NaCl, 1 mM EDTA, 10% glycerol, 1 mM DTT, 1 Roche tablet, 1 mM PMSF (Sigma), and DNAse) and lysed with Microfluidizer cell disruptor (Microfluidics) at 80 PSI. Cells were pelleted for 30 minutes at 34000 g. The supernatant was incubated with chitin resin (NEB) in a conical tube for 2 hr at 4 °C to favour binding (batch binding), then loaded on a column. The column was washed with 200 ml HEGX buffer, after which 12 ml of TGEX buffer with 100 mM DTT was loaded onto the column and 5.5 ml was drained out of the column. The column was left closed at 4 °C for 48 hr to ensure cleavage of the Tn5 from the intein. Elution was done in 1 ml aliquots and the concentration of the protein was measured via Bradford assay. The protein was concentrated with centrifugal filter units 10 K (Millipore). The activity of the protein was measured according to Picelli et al.,^{29 (link)}.

All chemicals and reagents were purchased from Sigma unless stated otherwise. The PCR reagents, restriction enzymes and B-PER reagent were obtained from Thermo Scientific. The cysteine-alkyne bifunctional linker (Figure S1A, Supplementary Materials) was purchased from Eurogentec, the azido-propylamine linker from Jena bioscience (Figure S1B, Supplementary Materials) and the N-hydroxysuccinimide (NHS) derived ester linker 2,5-dioxopyrrolidin-1-ylhex-5-ynoate (Figure S1C, Supplementary Materials) was self-synthesized according to Jagadish et al. [55 (link)]. The pMXB10 vector, E. coli SHuffle^® T7 competent cells and chitin resin were purchased from New England Biolabs. The two recombinant human VCAM1 antigens (hVCAM1) were bought from R & D Systems (MW of 270 kDa) and Peprotech (MW of 180 kDa) while the recombinant mouse VCAM1 (mVCAM1) antigen was purchased from Bioconnect (Huissen, The Netherlands, MW of 95 kDa). The ellipsometer and silicon wafers were bought from Synapse B.V. (Maastricht, The Netherlands) and the Biacore™ (Diegem, Belgium) C1 sensor chips from GEHealthcare. The SPR experiments were performed with a Biacore™ T200 model (GE Healthcare).