Mouse anti dnak

Samples were subjected to SDS-PAGE and transferred to nitrocellulose or PVDF membranes. Blots were blocked for 1 h in 5% (w/v) skim milk-PBST (0.1% Tween 20 in PBS) and then incubated with the primary antibody diluted in 5% skim milk-PBST for 1 h at room temperature. The optimal dilution for each antibody was calibrated individually. The secondary antibodies were diluted in 5% skim milk-PBST, incubated with the blots for 1 h at room temperature and detected with ECL reagents. The following commercial antibodies were used: mouse anti-DnaK (Abcam), mouse anti-JNK (BD Pharmingen), and mouse anti-actin (MPBio). Antibodies directed against T3SS components were a generous gift from Prof. B. Brett Finlay (University of British Columbia, Canada) and included mouse anti-EspA, mouse anti-EspB, rat anti-EscJ, mouse anti-Tir, and rabbit anti-SigD. Secondary antibodies were HRP-goat anti-mouse (Abcam) and HRP-goat anti-rat (Jackson ImmunoResearch).

Bacterial culture and total protein preparation as described “AmpC β-lactamase activity assay” and 30 μg total protein was taken for carrying out western blot assay. For CFZ and IMP treatment assays, the reagents were employed at the sub-culture stage. Western blot analysis was carried out using the standard methodology as elaborated earlier [36 (link)]. The detailed attributes of antibodies employed in western blot assay are as follows: rabbit anti-NagZ (preparation by ourselves), mouse anti-DnaK (Abcam, Cambridge, MA, USA), rabbit anti-AmpC (Abnova Taipei, Taiwan, China), goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). SPOT-CCD camera was used to take images. Software image J was employed for the quantification of the intensity of protein bands and DnaK was used as the internal control.

Samples were subjected to SDS-PAGE and transferred to nitrocellulose membrane (pore size, 0.45 µm; Bio-Rad). The membranes were blocked for 1 h with 5% (w/v) skim milk/PBST (0.1% Tween in phosphate-buffered saline), incubated with the primary antibody (diluted in 5% skim milk/PBST) for 1 h at room temperature or overnight at 4°C, washed, and then incubated with the secondary antibody (diluted in 5% skim milk/PBST) for 1 h at room temperature. Chemiluminescence was detected with EZ-ECL reagents (Biological Industries). The following primary antibodies were used: mouse anti-His (Pierce), diluted 1:2,000; rabbit anti-MBP (Thermo Fisher Scientific), diluted 1:1,000; mouse anti-DnaK (Abcam, Inc.), diluted 1:5,000; and mouse anti-actin (MPBio), diluted 1:10,000. Antibodies directed against T3SS components were a generous gift from Prof. B. Brett Finlay (University of British Columbia, Canada) and Prof. Rebekeh DeVinney (University of Calgary, Canada) and included mouse anti-Tir, rat anti-Intimin, and rat anti-Tir. Horseradish peroxidase-conjugated (HRP)-goat anti-mouse (Abcam Inc.), HRP-conjugated goat anti-rabbit (Abcam Inc.), and HRP-conjugated goat anti-rat (Jackson ImmunoResearch), diluted 1:10,000, were used as the secondary antibodies. Representative western blots of at least three independent experiments are presented in the Results section.