Versadoc mp 4000 imaging system

Human platelets were incubated with mAb (NIT A, NIT B or NIT F) and DANA or 10 μM PP1, or 10 μM SB253580 as described above and lysed in 1 × Laemmli buffer. For detection of phospho-p38MAPK, 1 × Halt protease inhibitor cocktail (Thermo Scientific) was also added. Samples were reduced with addition of 0.1 M dithiothreitol and boiled at 95^°C for 10 min. Platelet lysate was separated by SDS–polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membrane (GE Healthcare). For RCA-1 staining, membranes were probed with biotin-conjugated RCA-1 (2 μg ml⁻¹) (Vector Labs) according to the manufacturer's instructions, mouse anti-human CD41b antibody (1:5,000) (clone EPR6995, Abcam) and with β-actin antibody (1:400) (clone C1, Santa Cruz). For phospho-p38MAPK, membranes were probed with phospho-p38MAPK (Thr180/Tyr182) antibody (1:5,000) (Cell Signaling Techonology) at 4 °C overnight. Avidin conjugation to RCA-1 was performed with a Vectastain ABC kit (Vector Labs) according to the manufacturer's instructions. HRP-conjugated secondary anti-mouse antibody (1:5,000) (Santa Cruz) was then utilized. Blots were developed by ECL (Thermo Scientific) and visualized on the VersaDoc MP 4000 imaging system (Bio-Rad). Uncropped immunoblots are shown in Supplementary Fig. 9.

Here, we briefly summarise the 2-DE procedure as the procedure was described in detail earlier [10 (link),23 (link),50 (link)]. Proteomic analysis of depleted plasma samples and prepared muscle biopsies was carried out using Ettan IPGphor 3 IEF System (GE Healthcare, Buckinghamshire, UK) (first dimension) and Ettan DALTsix Electrophoresis Unit (Amersham, Pharmacia, Uppsala, Sweden) (second dimension). The fluorescent stain SYPRO Ruby (Bio-Rad Laboratories, Hercules, CA, USA) was applied to plasma protein gels, and silver stain was applied to muscle biopsy gels. The stained protein pattern was visualized using a charge coupled device camera (VersaDoc Imaging system 4000 MP, Bio-Rad). The PDQuest Advanced (v. 8.0.1, Bio-Rad) software was used to analyse and quantify the protein pattern. The amount of protein in a certain spot was assessed as background corrected optical density integrated over all pixels in the spot and expressed as integrated optical density (IOD). The parts per million (ppm) values for all proteins were generated and used for further statistical analysis.

Plasmid DNA was extracted from KPC-2 and KPC-17 KPs with the Qiagen Midi Kit (Qiagen). HindIII digested plasmids were separated in 0.8% agarose and transferred to nylon membranes for Southern hybridization. KPC-containing fragments were identified by hybridization with Dig-labeled bla_KPC-specific probe generated by the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche Applied Sciences, Germany). The images were visualized by using the VersaDoc Imaging System 4000MP (Bio-Rad, USA).
The extracted KPC-2- and KPC-17-producing plasmids described above were also transformed into NEB 10-beta electrocompetent E.coli recipients (C3020K, BioLabs) by using the Gene Pulser Xcell™ electroporation system (Bio-Rad). Transformants were confirmed to have bla_kpc-2 or bla_KPC-17 by PCR and sequencing analysis. Extracted plasmid DNA from the transformants was digested with HindIII and separated in a 0.8% agarose gel. The agarose gel was stained with ethidium bromide and visualized by using the Gel Doc™ XR+ system (Bio-Rad, USA).

To examine
the yeast cell-killing efficacy of the generated NFAP γ-core
peptide derivatives, the growth ability of C. albicans SC5314 was monitored in the presence of 0.5× MIC, 1× MIC,
and 2× MIC of the peptides that proved to be effective in the
antifungal susceptibility tests. The experiment was conducted in a
flat-bottom 96-well microtiter plate (TC Plate 96 Well, Suspension,
F; Sarstedt, Nümbrecht, Germany): 100 μL of OD₆₀₀ = 0.2 mid-log phase cells prepared in LCM were mixed with 100 μL
of 1× MIC, 2× MIC, or 4× MIC of the peptide diluted
in LCM. The plates were incubated statically at 30 °C for 24
h. The absorbance (OD₆₀₀) of each well was then measured
after shaking for 5 s every hour using a microtiter plate reader (BioTek
Synergy HTX Multi-Mode Microplate Reader; Agilent Technologies, Santa
Clara, CA, USA). Untreated cells (100 μL LCM mixed with 100
μL OD₆₀₀ = 0.2 mid-log phase cells prepared in LCM)
served as growth controls. To examine the viability of the treated C. albicans cells, 5–5 μL cultures from
each well were dropped onto the YPD agar plate after the last OD₆₀₀ measurement and allowed to dry before incubation at 30
°C for 24 h. The plates were photographed (Versa Doc Imaging
System 4000 MP; Bio-Rad, Hercules, CA, USA). These experiments were
repeated two times, involving three technical replicates.