Imagequant las 4000 mini chemical luminescent imager

To analyze the protein translation levels, the Western blot assay was performed as previously described [37 (link)]. DPS-1 was grown at an OD_600nm of 0.6 in MHB and treated with graded sub-inhibitory concentrations (1/2 × MIC, 1/4 × MIC, 1/8 × MIC, and 1/16 × MIC) of SKN. After shaking the culture, the bacterial cells were collected by centrifuging at 13,000 rpm for 10 min and lysed with SMART™ bacterial protein extraction solution (Intron Biotechnology, Seongnam, Korea). The suspension was centrifuged at 13,000 rpm for 10 min to obtain the soluble protein. The amount of protein was quantified using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, CA, USA). An equal number of sample proteins were loaded for 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) blotting membranes. After blotting, the membranes were blocked with 5% skim milk for 2 h and subsequently probed with the primary antibody overnight at 4 °C. The loading differences were normalized with a mouse monoclonal anti-GAPDH antibody. After incubating with the secondary antibody for 1 h, TOPview™ ECL substrate (Enzynomics, Daejeon, Korea) was supplemented to the membranes, and the immunoreactive protein bands were visualized using the ImageQuant LAS-4000 mini chemical luminescent imager (GE Healthcare Life, Seoul, Korea).

The western blot assay was performed according to the manufacturer’s instructions to analyze protein translation levels. The MRSA (ATCC 33591) suspensions (OD_600nm value of 0.7) were treated with graded sub-inhibitory concentrations of DMC and GEN. After shaking culture, the bacterial cells were harvested and suspended in bacterial protein extraction solution. Soluble protein was obtained by centrifuging the bacterial lysates at 13,000 rpm for 10 min. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to separate denatured protein (20 μL). The electrophoresed gels were transferred onto polyvinylidene difluoride (PVDF) blotting membranes. The membranes were blocked in 5% skim milk for 2 h, and then incubated with first antibody (anti-PBP2a and anti-SEB) overnight at 4 °C. After incubation with secondary antibody for 1 h, the immunoreactive proteins were detected by TOPview^TM ECL Femto Western Substrate (Enzynomics, Korea). ImageQuant LAS-4000 mini chemical luminescent imager (GE Healthcare Life, Korea) was used to visualize the immunoreactive protein bands of membranes.