Ai600 imager

For immunological detection of the HemR1 and HemR1′-′GFP proteins, Y. enterocolitica strains were grown under the desired conditions and extracts prepared from equal numbers of cells. The protein concentrations in the extracts were determined using the RC-DC protein assay (Bio-Rad) and if necessary they were diluted in Laemmli buffer to achieve equal loading (Sambrook and Russell, 2001 ). Samples were separated by electrophoresis on 12% TGX Stain-Free FastCast Acrylamide gels. Each gel was then Stain-Free activated by a 5 min exposure to UV and imaged using a GE Healthcare AI600 Imager. In some experiments, the loading of equivalent amounts of protein was controlled by Coomassie blue staining of an identical gel run in parallel. Next, the proteins were transferred to nitrocellulose membrane (Amersham Protran Western blotting membrane, nitrocellulose, pore size 0.2 μM; GE Healthcare) using a wet electroblotting system (Bio-Rad). The blots were probed with polyclonal rabbit antibodies against HemR1 (1:10,000, generous gift of Jürgen Heesemann) or GFP (Sigma Aldrich, 1:10,000). Sheep anti-rabbit IgG, conjugated to horseradish peroxidase (HRP, Promega) was used as the secondary antibody (1:15,000). Positive immunoreaction was visualized using Clarity ECL Blotting Substrate (Bio-Rad) for HRP-based chemiluminescent detection (GE Healthcare AI600 Imager).

Overproduction and purification of OmpR-His₆ and its use in an EMSA to detect interactions of this recombinant protein with DNA sequences were performed as described previously [64 (link)]. Briefly, a 255-bp fragment of the promoter region of the lcrGVsycD-yopBD operon and a 304-bp 16S rDNA fragment as a nonspecific competitor were PCR-amplified using Y. enterocolitica genomic DNA as a template and the pairs of primers listed in Table S1. The DNA fragments were incubated in OmpR-binding buffer (50 mM Tris-HCl pH 8.0, 100 mM KCl, 1 mM EDTA, 1 mM DTT, 20 mM MgCl₂, 12% glycerol, 100 µg/mL BSA, 0.1% Triton X-100) with increasing amounts of OmpR-His₆ (molar ratio of 1:20–300) at 26 °C for 30 min. The samples were then loaded on a 4.2% native polyacrylamide gel (19:1 acrylamide/bisacrylamide, 0.2 × TBE) containing 2% glycerol and electrophoresis performed at 110 V for approximately 3 h. The gel was then stained in 1 × SYBRgreen solution (Invitrogen, Waltham, MA, USA) and visualized using a GE Healthcare AI600 imager (GE Healthcare, Chicago, IL, USA).

The cells and tumor tissues were lysed using RIPA buffer (Beyotime) with protease and phosphatase inhibitors (Roche). Total protein concentration was estimated using the BCA quantitative assay (Protein Technology). Approximately 10 ug of protein samples was resolved using SDS-PAGE and transferred onto 0.2 μm PVDF membranes (Millipore). Membranes were blocked by 5% skim milk in TBST for 1 h and probed with primary antibodies against YBX3 (Immuno-Biological Laboratory); GAPDH, AKT, phospho-AKT, E-cadherin, Vimentin, and MMP1 (Protein Technology); PI3K and phosphor-PI3K (Cell Signaling Technology) at 1:1,000 dilution at 4°C overnight, washed with TBST three times, and then incubated with secondary antibodies against Anti-mouse IgG and Anti-rabbit IgG HRB-linked antibody (Cell Signaling Technology) at 1:2,000 dilution for 1 h at 25°C. Next, the specific protein bands were visualized using Chemiluminescence ECL kit (Millipore) and detected by GE Healthcare AI600 Imager (General Electric Company).