Hybond p

Bacteria pellets were resuspended in PBS with 1X Final Sample Buffer (FSB) and, after boiling at 100 °C, loaded on 12.5% SDS-PAGE. A protein molecular weight marker (Page ruler; Thermo Fisher) was included in each electrophoresis run. Proteins were transferred to nitrocellulose membranes (Hybond-P, Millipore), and the immunoblotting was carried out with Monoclonal ANTI-FLAG® M2 antibody (Sigma-Aldrich) and rabbit polyclonal anti-OmpA^{53 (link),54 (link)} as primary antibodies, and HRP conjugated goat anti-mouse and anti-rabbit IgG antibody as the secondary antibodies (Sigma-Aldrich). Signals were produced with ECL Star (Euroclone) and detected with ChemiDoc™ Gel Imaging System (Bio-Rad Laboratories). The densitometric analysis was performed by ImageJ software^{55 (link)}.

Whole cell extracts (WCEs) were prepared by lysing exponentially grown bacteria in 1X Laemmli buffer (Laemmli, 1970 (link)). In parallel, the same bacterial cultures were washed twice with PBS5 and OMPs were extracted as described previously (Cuenca et al., 2003 (link)). Briefly, bacterial cells were sonicated, treated with N-lauryl-sarcosine and resuspended in Cracking Dye (2% SDS, 20% glycerol, 62.5 mM Tris-HCl pH 6.8, 0.05% bromophenol blue, and 5% β-mercaptoethanol); WCEs were denatured for 10 min whereas OMPs for 5 min. Proteins were resolved by 12.5% Tris-glycine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and stained with Coomassie brilliant blue R-250 (Sigma) or electrotransferred onto PVDF membranes (Hybond-P, Millipore). Blots were probed with A. baumannii anti-OmpA serum (a kind gift of Prof. Smani) and with anti-phosphorylcholine (ChoP) TEPC 15 monoclonal antibody (Sigma), and an anti-mouse secondary antibodies IgG and IgA conjugated to horseradish peroxidase, respectively (Bio-Rad). Blots were visualized by enhanced chemiluminescence system (GE-Healthcare Bio-Sciences). From the Coomassie-stained SDS-PAGE, four gel slices were excised from each lane and subjected to in-gel digestion with trypsin, as previously described (Di Francesco et al., 2012 (link)). The tryptic peptides were extracted and dried in a speed vacuum.

The expression of proteins related with the adipogenesis and inflammation was measured by western blotting and according Grancieri, Martino and Gonzalez de Mejia [25 (link)]. Cell culture were collected after each treatment and lysed with RIPA lysis buffer, sonicated and added with Laemmli buffer (Bio-Rad) containing 5% β-mercaptoethanol, then the cell lysed were frozen at −80 °C. Protein concentration was quantified using RC-DC Assay (Bio-Rad) and 20 µg loaded in 4–20% Tris−HCl gels (Bio-Rad) for protein separation. Proteins were transferred to a PVDF membrane (polyvinylidene difluoride membrane, Hybond-P, Millipore, Billerica, MA, USA) and incubated overnight with respective primary antibodies (1:500) (COX-2, p65-NF, PPARγ, FAS, LPL or SREBP1) at 4 °C. The membranes were incubated with secondary antibody for 2 h (if required) and the protein bands visualized with a GL 4000 Pro Imaging system (Carestream Health Inc., Rochester, NY, USA). Band intensity was normalized using GAPDH antibody. All analyses were performed in duplicate and results expressed in % of expression compared to positive control.

Immunoblotting was as described (12 (link)). Briefly, cells were seeded, treated as required and protein was harvested using RIPA buffer and a cocktail of protease/phosphates inhibitors (#A32963, Thermo Scientific). Protein was quantified using the BCA assay (#23227, Thermo Fisher Scientific) and 20-μg protein was resolved on appropriate SDS-polyacrylamide gels depending on the size of the protein of interest. Proteins were transferred onto Hybond-P (#GE10600023, Sigma) and probed using appropriate antibodies (see Supplementary Methods). Immunoreactivity was detected using the SuperSignal West Pico Detection Kit (#34577, Thermo Fisher Scientific). The level of protein signal was quantified using Image J.