Co-immunoprecipitation (Co-IP) The GVE2-infected Geobacillus sp. E263 was collected by centrifugation at 7,000× g for 10 min. The precipitate was re-suspended in 0.1 M Tris–HCl (pH 7.5). After sonication for 5 min, the learn more suspension was centrifuged at 12,000×g for 15 min. The appropriate immunoprecipitation antibody was added to the supernatant and incubated for 2 h at 4°C. Protein A Sepharose slurry (Bio-Rad) was subsequently added, followed by incubation for 2 h at 4°C.

Nonspecific binding proteins were removed by five successive rinses with phosphate buffered saline (PBS). The Protein A Sepharose was finally selleck compound eluted with glycine solution (0.1 M; pH 1.8). The eluant was collected and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Mass spectrometry (MS) analysis The protein bands of the SDS-PAGE were excised, trypsinyzed and analyzed using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) MS. A 1.5-μL aliquot was spotted onto a MALDI-TOF sample plate with an equal volume of matrix, a saturated solution of α-cyano-4-hydroxycinnamic acid (Sigma, USA) in 0.1% trifluoroacetic

acid and 50% acrylonitrile. The samples were analyzed using a Bruker AutoFlex MALDI-TOF mass spectrometer (Bruker Daltonics, USA). All peptide mass finger printings were externally calibrated using standard peptide mixtures and internally calibrated using the masses of trypsin autolysis products to reach a typical mass measurement accuracy of 100 ppm. All acquired sample spectra were processed www.selleckchem.com/products/GDC-0941.html using Bruker Flexcontrol 2.4 operation software (Bruker Daltonics) in a default mode with an MS tolerance of 0.2 Da and a tandem MS tolerance of 0.6 Da. Protein identification was performed using Mascot software (version 2.1; Matrix Science, London, UK) and GPS Explorer software (version 3.6; Applied Biosystems, USA) against the NCBInr database and the ORF database of Geobacillus

kaustophilus HTA426 in a local database that was generated using a shotgun approach. To eliminate protein redundancy in the database under different names and accession Inositol oxygenase numbers, the single protein member belonging to the species G. kaustophilus HTA426 or otherwise had the highest protein score (top rank) was singled out from the multi-protein family. Northern blot analysis Total RNAs were respectively isolated from thermophilic Geobacillus sp. E263 before and after GVE2 infection using Trizol reagent (Invitrogen, USA), followed by incubation with RNase-free DNase I (TakaRa, Japan) for 30 min at 37°C. After electrophoresis on a 1.2% agarose gel in 1× Tris-borate-ethylenediaminetetraacetic acid buffer, the RNAs were transferred to a nylon membrane (Amersham Biosciences, USA). The blots were probed with digoxigenin (DIG)-labeled vp371, GroEL, or AST, respectively. Bacterial 16S rRNA gene was used as a control.