Amersham protran 0.45 μm nitrocellulose membrane

Western blot was performed on triplicate infections carried out in confluent HFF in 6-well plates at the indicated MOI and treatment conditions. Sonicated whole-cell extracts in lysis buffer containing phosphatase and protease inhibitors were prepared as previously described (44 (link)). The gel-loading buffer contained 2% SDS and 100 mM beta-mercaptoethanol. The proteins were fractionated on freshly made 8% SDS-PAGE Tris-glycine gels prior to transfer to Amersham Protran 0.45-μm nitrocellulose membranes (GE Healthcare Life Sciences 10600002). HCMV IE1-72 and IE2-86 were detected by murine monoclonal antibody MAB810 (EMD Millipore, 1:1,000 dilution). Host actin was detected with polyclonal rabbit anti-actin antibody (Sigma-Aldrich, A2066, 1:4,000 dilution). Primary antibodies were detected with peroxidase AffiniPure F(ab’)2 fragment goat anti mouse IgG (Jackson ImmunoResearch, 115-036-006) at 1:40,000 dilution and goat anti-rabbit IgG (whole molecule)–peroxidase antibody (Sigma-Aldrich, A0545) at 1:40,000 dilution. Blots were imaged and analyzed using the iBright FL1500 Imaging System (Invitrogen).

After electrophoresis (SDS–PAGE, or Phos‐tag SDS–PAGE), proteins were transferred onto Amersham™ Protran™ 0.45 μm nitrocellulose membranes (GE Healthcare) or onto Amersham™ Hybond ™ P 0.45 μm PVDF membranes (GE Healthcare). Phosphorylated Hog1 was detected by immunoblotting using the anti‐phospho‐p38 MAPK (T180/Y182) antibody #9211 (Cell Signaling Technology). Hog1 was detected using the anti‐Hog1 antibody yC‐20 (Santa Cruz Biotechnology). FLAG‐Hog1 was detected by anti‐FLAG M2 antibody (Sigma‐Aldrich). Pbs2‐HA was detected by anti‐HA antibody F‐7 (Santa Cruz Biotechnology). Phosphorylation of Pbs2 Thr‐518 was detected by polyclonal anti‐Pbs2 phospho‐T518 antibody custom produced by SCRUM Inc. Phosphorylated Kss1 and Fus3 were detected using the anti‐phospho‐p44/42 MAPK (Erk1/2; T202/Y204) Rabbit monoclonal antibody #4370 (Cell Signaling Technology).

Extracellular proteins from the supernatant of SXM cultures were precipitated with 10% TCA (w/v) in acetone at 4°C overnight. This mixture was centrifuged at 4 000 rpm for 60 min at 4°C. The protein sediment was washed three times with 80% (v/v) acetone, once with 100% (v/v) acetone and then dissolved in 8 M urea/2 M thiourea. Intracellular proteins were extracted from ground mycelium with extraction buffer [300 mM NaCl, 100 mM Tris−HCl pH 7.5, 10% glycerol, 1 mM EDTA, 0.02% NP-40, 2 mM DTT and complete EDTA-free protease inhibitor cocktail (Roche)]. Samples were centrifuged for 20 min at 13 000 rpm at 4°C and the supernatants were transferred into fresh test tubes. Protein concentrations were determined using a Bradford-based Roti-Quant assay (Carl Roth). Protein samples were separated in 12% SDS−PAGE gels, followed by protein transfer onto an Amersham Protran 0.45 μm nitrocellulose membrane (GE Healthcare). The membrane was blocked in 5% (w/v) skim milk powder in TBS-T [10 mM Tris−HCl (pH 8), 150 mM NaCl, 0.05% (w/v) Tween 20] and probed with α-GFP antibody (Santa Cruz Biotechnology). As secondary antibody the horseradish peroxidase-coupled α-mouse antibody (115-035-003, Jackson ImmunoResearch) was applied. Detection of chemiluminescent signals was conducted with horseradish peroxidase substrate luminol based chemiluminescence.

Secretion of type III effector ExoS can be induced in vitro by removing calcium from the medium [29 (link)]. A secretion assay was performed as previously described [29 (link)] with a slight modification. Bacteria were diluted 1:300 into “high-salt” LB (medium containing 200 mM NaCl, 10 mM MgCl₂, and 0.5 mM CaCl₂) and grown for 2 h, at which point secretion was induced through the addition of EGTA (5 mM, final concentration). The cultures were allowed to grow for another 2 h, and the bacteria were pelleted by centrifugation. As required, _L-serine was added at the indicated concentrations to the culture. Supernatant proteins were precipitated with 10% (final concentration) trichloroacetic acid (TCA). Pellets were washed three times with acetone and resuspended in phosphate buffered saline (PBS). After determining the protein concentration with a BCA Protein Assay Reagent kit (Thermo Fisher Scientific), samples (8 μg protein) were separated by SDS-PAGE, transferred to an Amersham Protran 0.45-μm nitrocellulose membrane (GE Healthcare), and probed with a rabbit anti-ExoS polyclonal antibody. Anti-ExoS polyclonal antibody was prepared as previously described [30 ].