Hrp conjugated donkey anti rabbit antibody

Total membranes were prepared to determine the expression levels of S1PRs. Colon samples were homogenized on ice for 3 minutes in 20 mM Tris-HCl, pH 7.5 containing 2 mM MgCl₂, 0.25 M sucrose, and 1x protease inhibitors (Sigma Aldrich, Munich, Germany). Homogenates were centrifuged at 3000 x g for 10 minutes at 4°C and supernatants were subjected to another centrifugation step at 100,000 x g for 1 hour at 4°C. The pellet was suspended in 50 mM Tris-HCl, pH 7.5 containing 1x protease inhibitors cocktail (Sigma Aldrich, Munich, Germany) and 0.1% sodium dodecyle sulfate. Protein concentration was measured using the BCA-protein determination kit (Themoscientific, Ottawa, Ontario, CA) and samples were stored at -80°C for further analysis.
After boiling, samples from control and colitic colon segments (50 μg protein) were subjected to SDS-PAGE as previously described [44 (link)]. The membranes were immunoblotted with rabbit anti-S1PR1 (Cayman Chemical, MI, USA), rabbit anti-S1PR2 (Sigma Aldrich, Munich, Germany), rabbit anti-S1PR3 (Cayman Chemicals, MI, USA) or rabbit anti-actin (Abcam, MA, USA). HRP-conjugated donkey anti-rabbit antibody (Jackson ImmunoResearch, PA, USA) was used as secondary antibody. Bands were detected using Super Signal Western Pico chemiluminescence Substrate (Thermoscientific, Ottawa, Ontario, CA) and quantified using Image J software.

Protein samples were separated by SDS–polyacrylamide gel electrophoresis and blotted on polyvinylidenfluoride membranes (Merck Millipore). Membranes were blocked overnight with Tris-buffered saline 1X (TBS) (50 mM Tris-HCl, 150 mM NaCl, pH 8) containing, 0.1% Tween-20 and 8% dry milk and then incubated for an additional 3 hr with the primary antibodies diluted in TBS 1X, 0.1% Tween-20, 5% dry milk. The different polyclonal antisera to ZitP (NTER, 1:5000 dilution and CTER, 1:5000), CtrA (1:10,000) and DivJ (1:10,000) were used. Commercial and polyclonal antibodies to Dendra2 (Antibodies-Online) and mCherry (Chen et al., 2005 (link)) were used at 1:5,000 and 1:10,000 dilutions respectively. Primary antibodies were detected using HRP-conjugated donkey anti-rabbit antibody (Jackson ImmunoResearch) with ECL Western Blotting Detection System (GE Healthcare) and a luminescent image analyzer (Chemidoc^Tm MP, Biorad).

The appropriate strain was grown for 2 to 4 hours at 30°C under constant agitation up to OD_600nm of 0.4 to 0.6, then the inducer was added, and the cells were grown for 2 additional hours. For chase experiment, the strain was grown similarly up to OD_600nm of 0.3 to 0.5, then chloramphenicol 5 μg/mL was supplemented with vigorous mixing, immediately followed by the addition of mentioned inducer. Protein samples from exponentially growing cells were separated on a SDS–polyacrylamide (37.5:1) gel electrophoresis and blotted on 0.45 μm pore PolyVinyliDenFluoride (PVDF) membranes (Immobilon-P from Sigma Aldrich). Membranes were blocked for 2 hours with 1× Tris-buffered saline (TBS) (50 mM Tris-HCl, 150 mM NaCl [pH 8]) that contain 0.1% Tween-20% and 8% powdered milk, followed by overnight incubation with the primary antibodies diluted in the same milk solution. The polyclonal antisera to AcrA (1:15,000 dilution), TipR (1:5,000 dilution), and CCNA_00164 (1:20,000 dilution) were used. The detection of primary antibodies was done using HRP-conjugated donkey anti-rabbit antibody (Jackson ImmunoResearch) with Western Blotting Detection System (Immobilon from Milipore), and an imaging was performed in Bio-Rad illuminator (Chemidoc MP, Bio-Rad).