Chemostar touch imager

To analyze protein expression, cells were lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, pH 7.2) supplemented with protease (Sigma) and phosphatase (Roche) inhibitors. Up to 75 μg of protein were separated by SDS-PAGE, blotted onto nitrocellulose and incubated overnight at 4 °C with primary antibodies (Table S2). Membranes were incubated with HRP-coupled secondary antibodies (santa cruz, CST) for 1 h at RT and chemiluminescence detected with ECL Prime (GE Healthcare) at the LAS-1000 (Fujifilm), Amersham Imager 600 (GE Healthcare) or ChemoStar Touch Imager (Intas). Image J software was used for densitometric quantification. For co-immunoprecipitation, cells were transfected with V5-tagged WNT11 and 24 h post transfection proteins were crosslinked for 30 min with 1 mM DSS (Thermo Fisher) in 1 ml PBS + 1 mM MgCl₂. Cells were lyzed in 50 mM Tris/HCl pH 8.0, 150 mM NaCl, 1% NP-40 + protease inhibitors. Five hundred micrograms protein were incubated with 1 μg normal rabbit IgG (#2729) or V5 antibody (#13202, both CST) for 16 h at 4 °C. Antibody-protein complexes were incubated for 2 h at 4 °C with protein A/G agarose beads (#sc-2003, santa cruz) and spun down at 1700 g for 1 min. Signals were visualized by western blot as described above using the confirmation-specific mouse anti-rabbit IgG (#3678, CST) for V5 detection.

Overnight cultures of Salmonella SB905, the SpaQ and SpaR mutants or the complemented SpaP-knockout strains were grown at 37 °C in 5 ml of LB medium containing 0.3 M NaCl and antibiotics. The next morning, cultures were diluted 1:10 in 50 ml of the same medium without antibiotics. The expression of HilA was induced by addition of 0.012% w/v l-arabinose for another growth period of 5 h. Afterwards, the cell density was adjusted to an OD₆₀₀ of 1.0 and the cells were pelleted at 3146 × g for 10 min. Supernatants were collected and filtered with a 0.22 μM syringe filter. Pellets were resuspended in 1× phosphate-buffered saline (PBS). Both supernatants and cell pellets were then immunoblotted with antibodies raised against effectors SptP and SipA or needle complex proteins (generated in-house, 1:5000 dilution for anti-SptP and 1:10,000 dilution for anti-SipA and anti-needle complex), and then secondary anti-rabbit or anti-mouse horseradish peroxidase (HRP)-conjugated antibodies (Sigma Aldrich/Merck kGaA; 1:10,000 dilution each). Immunoblots were imaged using Intas Science Imaging Instruments GmbH ChemoStar Touch imager running v.0.5.65 software and were quantified using ImageJ v.1.50i software^{35 (link)}.

40 μg protein/lane were loaded on a 11% polyacrylamide gel. Proteins were transferred to nitrocellulose membrane (GE HealthCare, Freiburg, Germany). Membranes were washed in TBST (TBS with 1% Tween 20), blocked for 1 h in 3% bovine serum albumin (BSA, Carl Roth, Karlsruhe, Germany) and incubated in MAb4a (1:500; 146,011, RRID:AB_887722, Synaptic Systems, Göttingen, Germany), anti-gephyrin (1:1,000; 147,111, RRID:AB_887719, Synaptic Systems, Göttingen, Germany), anti-GFP (1:5,000; SC8334, RRID:AB_641123, Santa Cruz Biotechnology, Dallas, TX, United States) or anti-pan-cadherin antibody (1:1000; 4,068, RRID:AB_2158565, Cell Signaling, Danvers, MA, United States) overnight at 4°C. As secondary antibodies horse radish peroxidase (HRP) conjugated goat anti-mouse (1:15,000, 115–035-146, RRID:AB_2307392, Dianova, Hamburg, Germany) or goat anti-rabbit secondary antibodies (1:15,000, 111–036-003, RRID:AB_2337942, Dianova, Hamburg, Germany) were used. Signal detection was done using the SuperSignal West (Thermo Fisher Scientific, Waltham, MA, United States). Western Blot Images were taken using the Chemostar Touch Imager (Intas Science Imaging Instruments, Göttingen, Germany).