Silverxpress kit

Isolates were plated on CR-TSA from 15% glycerol stocks, and incubated at 37°C overnight. One colony from each isolate was streaked again onto CR-TSA and grown as above. A loop of each solid culture was then processed for pINV extraction as previously described [59 ]. DNA samples were run on 0.7% agarose gel at 1 V/cm voltage for approximately 16 hrs.
To activate secretion through the T3SS, bacteria were grown overnight at 37°C in TSB at 180 r.p.m., then sub-cultured to an OD₆₀₀ of 0.05 into 10mL fresh TSB grown in the same conditions until OD₆₀₀ ~ 1 was reached. Cultures were centrifuged and pellets were resuspended in PBS to an OD₆₀₀ ~ 5. T3SS secretion was induced by adding Congo red (final concentration, 0.02% w/v) followed by incubation at 37°C for 15 minutes. Bacteria were pelleted and supernatants containing secreted proteins were boiled in an equal volume of 2x SDS-PAGE loading buffer (0.1M Tris-HCl ph 6.8, 1.5% SDS, 20% glycerol, 25mM EDTA, 2% β-mercaptoethanol, 150 μg/mL bromophenol blue) at a 1:1 dilution before loaded on 10% SDS-PAGE gels. After electrophoresis, gels were silver stained using the SilverXpress Kit (Invitrogen LC6100) following manufacter’s protocol.

Strains were grown until OD₆₀₀ = 1. Cultures were centrifuged at 15,000 g for 10 min at 4 °C and supernatants from equivalent cell numbers were subjected to SDS-PAGE and Silver-stained (Silver Xpress kit, Invitrogen) or Western blotted.

Shigella flexneri strains (Table 1) were grown in TCSB at 37°C until A_600nm of ∼ 1.0, at which point samples were taken for Western blotting of whole cell lysate. The CR induction assay was then performed as described previously (Kenjale et al., 2005). Twenty microlitres of bacterial supernatant was separated by SDS‐PAGE and silver‐stained with the SilverXpress kit (Invitrogen).

Ricin, abrin, abrin A-chain, abrin B-chain at a concentration of 500 ng per well in the presence or absence of reducing agent (0.05 M DTT) were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) with NuPAGE 4–12% Bis-Tris gels (Invitrogen) followed by either Silver stain or Western blotting. One gel was subsequently silver stained using the SilverXpress kit according to the manufacturer’s instructions (Invitrogen). For the remaining gels, the resolved proteins were transferred to a PVDF membrane (Invitrogen iBlot). The membranes were blocked in 5% milk-Tris-buffered saline-0.1% Tween 20 buffer then probed with each of the 10 monoclonal LSABx antibodies at a concentration of 5 μg/mL in blocking solution followed by secondary antibody (goat anti-mouse horseradish peroxidase (HRP)-conjugated; 1:3000, Cell Signaling Technology). The blot was incubated in Pierce ECL Western Blotting Substrate solution (Thermo Scientific). Protein bands from peroxidase activities to chemiluminescent substrates were developed and detected using the ChemiDoc MP imaging system (BIO-RAD, Hercules, CA, USA). Molecular weight standards were purchased from Invitrogen.

Abrin, abrin A-chain, and abrin B-chain at the concentration of 100 ng per well in the presence or absence of a reducing agent (0.05 M DTT) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with NuPAGE 4–12% Bis-Tris gels (Invitrogen) followed by silver staining using the SilverXpress kit according to the manufacturer’s instructions (Invitrogen). The silver stained gel image was acquired using the ChemiDoc MP imaging system (BIO-RAD, Hercules, CA, USA). Molecular weight standards were purchased from Invitrogen. Abrin untreated or treated with either increasing temperatures or pH in 1× PBS with 0.2% phosphate buffer gelatin were loaded at 100 ng per well as above. Electrophoresis and silver-staining procedures were followed as stated.

BSR monolayers were infected with mutant BTV. Medium was discarded at 24 hpi and 0.1 ml/cm² Trizol was added to the cells and incubated for 5 min at room temperature. After harvesting, 0.2 ml chloroform/ml Trizol was added and the mixture was centrifuged for 10 min at 6,000 rpm. The water phase was isolated and 0.8 ml isopropanol/ml was added. Precipitated RNA was centrifuged for 30 min at 4°C and 13,000 rpm. The pellet was washed with 70% ethanol and dissolved in 100 μl RNase-free water. Fifty μl of 7M LiCl was added, followed by incubation for 30 min at –20°C to precipitate ssRNA. After centrifugation for 15 min at 4°C and 13,000 rpm, dsRNA was purified from the supernatant using the RNA clean and concentrator^tm-5 kit (Zymo research) according to manufacturer’s protocol. Approximately 200 ng dsRNA was separated by 4–12% polyacrylamide gel electrophoresis (PAGE) and visualized by silver staining using the SilverXpress kit (Invitrogen).