Amersham hybondtm p membranes

Western blot analysis was carried out to evaluate the effect of TCA on the expression of PBP2a according to the methods described previously [21 (link)]. S. aureus strains (ATCC 33591) were grown in MHB for 24 h and treated with sub-concentrations of TCA for 30 min. Cell protein extracts were harvested by centrifugation at 3000× g for 10 min and the protein concentration was determined by a Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). The supernatants were subjected to SDS-PAGE and electroblotted onto Amersham HybondTM-P membranes (GE Healthcare, Piscataway, NJ, USA). The membranes were blocked by 5% skim milk and probed with monoclonal mouse anti-PBP2a primary antibody (diluted 1:1000, DiNonA, Seoul, Korea) overnight at 4 °C and re-probed with anti-mouse IgG secondary antibody (diluted 1:2000, Enzo Life Sciences, Ann Arbor, MI, USA) at room temperature for 2 h. Then, the membranes were supplemented with ECL Prime Western Blotting Detection reagent (GE Healthcare Life Sciences, Incheon, Korea), and an ImageQuant LAS-4000 mini chemical luminescent imager (GE Healthcare Life Sciences) was used to visualize the bands [22 (link)].

Proteins from the cellular fraction of NPAs were extracted from QIAzol using chloroform-isopropanol and 1% SDS. Proteins were quantified by BCA (Thermo Fisher Scientific) and resolved (10 µg) in Acrylamide/Bis-acrylamide gels. They were then transferred to polyvinylidene difluoride (PVDF) Amersham HybondTM-P membranes (GE Healthcare, Buckinghamshire, UK) and blocked 2 h in 1× PBS/5% non-fat-dried milk/0.2% Tween-20 at room temperature. Overnight incubation was performed at 4 °C with antibodies against β-actin (Cell Signaling Technology Cat# 4970, RRID:AB_2223172, Leiden, The Netherlands, 1:1000) and filaggrin (Novus Biologicals, LLC, CO, USA, 1:500) in 1× PBS/0.5% non-fat-dried milk/0.2% Tween-20, and further incubation with the secondary anti-rabbit (Millipore Cat# AP156P, RRID:AB_11213985, Burlington, MA, USA, 1:1000) or anti-mouse (Thermo Fisher Scientific, 1:1000) antibody-HRP for 2 h at room temperature. Immobilon^® Crescendo Western HRP Substrate was used, with visualization using an Amersham Imager 600 (GE Healthcare). Data were curated using Quantity One 1-D software (Bio-Rad).

A Western blot assay was performed to evaluate the effect of BDMC on the expression of virulence-related exoproteins based on the previously reported investigation [21 (link)]. S. aureus strains (ATCC 33591) were cultured in MHB for 24 h and then treated for 4 h with sub-inhibitory concentrations of BDMC. Protein concentration was measured using a Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA) after centrifugation at 3000× g for 10 min to harvest cell protein extracts. The supernatant was separated by SDS-PAGE and electroblotted onto Amersham HybondTM-P membranes (GE Healthcare, Piscataway, NJ, USA) that had been blocked with 5% skim milk and probed for 8 h at 4 °C with anti-Staphylococcus alpha-hemolysin antibody, anti-Staphylococcus enterotoxin A antibody, and anti-Staphylococcus enterotoxin B antibody (diluted 1:1000), and then reprobed by anti-rabbit IgG secondary antibody (diluted 1:1000) at room temperature for 2 h. The membranes were then supplemented with ECL^™ Prime Western Blotting Detection reagent (GE Healthcare Life Sciences, Incheon, Korea). The bands were visualized using the ImageQuant LAS-4000 mini chemical luminescent imager (GE Healthcare Life Sciences).