Genesys software

For Coomassie blue staining, equal amounts of protein were resolved on a 4–20% Tris-glycine gel. The gel was stained with Coomassie blue for 2 h, destained overnight, and imaged on the InGenius 3 using the GeneSys software (SynGene). For immunoblotting, equal amounts of protein were resolved on a 3–8% Tris-acetate gel and transferred onto a nitrocellulose membrane for 90 min at 200 mA. The blots were blocked in 5% milk in Tris-buffered saline (TBS) for an hour and were incubated overnight with rabbit α-Matrin 3 antibody (1:10,000, Novus Biologicals, catalog # NB100-1761) or mouse α-GAPDH antibody (1:5,000, Meridian Life Sciences, catalog # H86504M) diluted in 5% milk/TBS. Following washes in TBS, the blots were incubated with horseradish peroxidase-conjugated α-rabbit or α-mouse for 1 h (1:5,000, Jackson ImmunoResearch Laboratories, Inc., rabbit catalog # 711-036-125, mouse catalog # 715-036-150). Following washes in TBS, membranes were incubated with ECL-Plus reagent (Fisher, catalog # 509049326) and imaged on PXi using the GeneSys software (SynGene).

Samples for Western blotting and immunoprecipitation were prepared under the same conditions as for the release assay, and analyses were performed essentially as reported [32 (link)]. Briefly, platelets (5 × 10⁸ cells/300 μl) were lysed and sonicated in sample buffer (10 mM Tris–HCl, 150 mM NaCl, 2% SDS, cOmplete, PhosStop). Equal amounts of protein were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membranes. The membranes were incubated with primary antibodies and then with secondary antibodies (DAKO; 1:10,000). Peroxidase activity was visualized with a chemiluminescent reagent (Immunostar: Wako) and G:Box apparatus (Syngene). Quantification was performed by GeneSys software (Syngene). For immunoprecipitation [32 (link)], platelets (5 × 10⁸ cells/300 μl) were lysed and sonicated in RIPA buffer (50 mM Tris/HCI, 150 mM NaCI, 1 mM EDTA, 5 mM EGTA, 1% NP-40, 20 mM glycerophosphate, 0.5 M DTT) containing cOmplete and PhosStop. After centrifugation, supernatants were preabsorbed with protein G-Sepharose (GE Healthcare). Supernatants were then incubated with anti-Syk antibodies overnight at 4 °C and the immunocomplexes were precipitated by the addition of 40 μl of Protein GSepharose for 2 h. After brief centrifugation, immunoprecipitates were washed 3 times with RIPA buffer and used for Western blotting.

DNA from cheeses and strains harbouring known tet genes (Lactococcus lactis IPLA 31008 [tet(M)], Enterococcus faecalis Jtet [tet(O)], Enterococcus spp. ET15 [tet(S)], and Bifidobacterium longum B93 [tet(W)]) was amplified by PCR using DGGE primers (Table 1), employing the PCR conditions described elsewhere [15 (link)]. DGGE analysis of the amplified tet genes was performed as previously reported [16 (link)] with slight modifications. Briefly, DGGE was performed in a DCode apparatus (Bio-Rad, Richmond, CA, USA) at 60°C on 8% polyacrylamide gels with a formamide-urea denaturing gradient of 15–50%. Electrophoresis was conducted at 150 V for 2 h and 200 V for 1 h. After electrophoresis, gels were stained in an ethidium bromide solution (0.5 μg mL⁻¹), and the DNA bands were visualized and captured using a Gbox system and GeneSys software (Syngene, Cambridge, UK). After isolation and reamplification with the same primers without the GC clamp and identical PCR conditions, bands from the acrylamide gels were identified by sequencing and sequence comparison. Online similarity searches were performed by using the BLAST tool in the GenBank database (http://www.ncbi.nlm.nih.gov/BLAST/).