Axygen

THP-1 cells, transduced to express various constructs of HA-US28, were lysed in radioimmunoprecipitation assay (RIPA) buffer, and nuclei and cell debris were removed by centrifugation at 13,000 × g for 10 min at 4°C. Proteins were separated on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Axygen; Corning). Incubations with primary and secondary antibodies were performed using 5% skimmed milk for 1 h each at room temperature. Proteins were detected using the following antibodies: anti-p42/p44 or phosphor-anti-p42/p44 antibodies or anti-MSK1 or anti-phosphor-MSK1 antibodies (serine 360) (all 1:1,000; Cell Signalling Technology, Danvers, MA) or anti-CREB or phosphor-CREB antibodies (S360) (both Merck). The secondary antibody used was chicken anti-rabbit horseradish peroxidase (Santa Cruz Biotech). Blots were developed with the use of enhanced chemiluminescence (GE Healthcare) and visualized with autoradiography film.
To detect cellular localization of NF-kB, cells were fractionated using REAP (rapid, efficient, and practical) (93 (link)) and proteins detected using the following antibodies: anti-NF-κB (Abcam, Inc.), anti-p84 (Thermo), and anti-GAPDH (Millipore). Secondary antibodies used were chicken anti-rabbit and bovine anti-mouse horseradish peroxidase (both Santa Cruz Biotech).

The protein band corresponding to PilS at the molecular weight of ~23 kDa was excised from the SDS-polyacrylamide gel and incubated for 3 h in a 50% methanol, 5% acetic acid solution to destain the gel and remove SDS. The supernatant was removed, and the gel was dehydrated with 100% acetonitrile followed by vacuum centrifugation (SpeedVac RVC-2-18/ Alpha 1-2, Christ, Osterode am Harz, Germany). To the dried gel fragment, 10 mM solution of dithiothreitol in 100 mM ammonium bicarbonate was added to reduce disulfide bridges for 30 min at room temperature, followed by incubation in the dark with 50 mM iodoacetamide in 100 mM ammonium bicarbonate for 30 min for alkylation of the SH groups of the cysteine side chains. The gel was washed with 100 mM ammonium bicarbonate and proteins were digested, incubating the gel overnight at 37 °C with 50 μg/mL of trypsin (Sigma-Aldrich, Saint Louis, MO, USA). The digested protein extraction was preceded by incubating the gel in 5% formic acid for 10 min and 5% formic acid in a 50% acetonitrile solution for 10 min. Supernatants were collected in a clean low-binding tube (Axygen, Corning Life Sciences, New York, NY, USA), vacuum dried and resuspended in 0.5% formic acid solution for mass spectrometry analysis.

A 10 mL blood sample was drawn from an antecubital vein in the forearm and collected into a serum vacutainer (BD Vacutainer systems, Plymouth, UK). The blood samples were obtained at each time point and left at 22–24 °C for 30 min to allow clotting, and then kept on ice when necessary. Serum samples were centrifuged at 1300g for 15 min at 4 °C. All samples were then aliquoted into 1.5 mL microcentrifuge tubes [Axygen (Corning), New York, USA] and stored at − 80 °C until subsequent analysis.