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 (r apid, e fficient, a nd p ractical) (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).
To detect cellular localization of NF-kB, cells were fractionated using REAP (