Nupage lds buffer

Western blotting was performed as previously described [31 (link)]. Briefly, equal mass aliquots of isolated cytoplasmic, nuclear, and input fractions were diluted with H₂O to 7.5 μl, mixed with 2.5 μl of NuPAGE LDS buffer (Invitrogen, catalog # NP0007), and heated to 70 °C for 10 min for denaturation. The isolates were separated on a NuPAGE 4–12% Bis-tris gel and transferred to a PVDF membrane. The primary antibodies used were mouse anti-tubulin alpha antibody (AA43) developed by Walsh, C (obtained from the Developmental Studies Hybridoma Bank at the University of Iowa, Department of Biology) and rabbit anti-histone H3 antibody (Novus Biologicals, catalog # NB500–171). The secondary antibodies used were goat anti-mouse IgG (H + L) antibody conjugated to HRP (Invitrogen, catalog # 31430) and goat anti-rabbit IgG (H + L) antibody conjugated to HRP (Promega, catalog # W4018). Immunoblots were imaged using iBright 1500 (ThermoFisher, catalog # A44241).

Total proteins were extracted by scraping and syringing cells in 1 × NuPAGE LDS buffer (Invitrogen). Fifteen to thirty micrograms of total proteins were then separated on 10% SDS-PAGE gels. The following antibodies were used: mouse monoclonal anti-Mdm2 (SMP14, sc-965, Santa Cruz Biotechnology or 2A10, MABE281# Merck Millipore), anti-p53 (DO-1, Santa-Cruz Biotechnology) and anti-NS1 (sc-130568, Santa Cruz biotechnology) antibodies, and a sheep polyclonal anti-p53 antibody (SAPU, JC Bourdon, University of Dundee). In addition, an anti-Ku80 polyclonal antibody was used as a loading control (#2753, Cell Signaling).

Cells were cultured on 8-well chamber slides for immunocytochemistry. Briefly, cells were fixed in either 4% formaldehyde or 1:1 methanol:acetone, blocked, and incubated with primary antibodies followed by Alexa fluor conjugated secondary antibodies or Alexa 488-conjugated phalloidin (for Actin staining). Chambers were then removed and slides mounted using Prolong Gold anti-fade with DAPI. Immunohistochemistry was performed on 5 µm thick paraffin embedded mouse lung sections. Following heat-induced antigen retrieval, sections were incubated with primary antibodies followed by Alexa conjugated secondary antibodies. Sections were mounted in Prolong Gold anti-fade with DAPI (Invitrogen). Images were acquired using either TissueFaxs (Tissugnostics), Nikon E800, Deltavision Eclipse, Olympus Fluoview confocal, or Zeiss confocal microscopes. Image analysis and intensity measurements were performed using ImageJ.
For protein gels, whole-cell lysates were prepared using RIPA buffer with protease and phosphatase inhibitors. They were then reduced in NuPage LDS buffer (Invitrogen), and a wet transfer was performed prior to western blotting. For histone blots, acid extracts were made following manufacturer (Abcam) recommendations. Refer to Key resource table for list of antibodies used.

HEK-293FT cells were chemically transfected (Lipofectamine 2000, Invitrogen) according to the manufacturers’ protocol. After 24 h, cells were lysed in 25 mM Tris–HCl pH 8.0, 150 mM NaCl, 1 × protease inhibitor cocktail (HALT), 250 U/mL benzonase (Invitrogen), 10 mM DTT, 1% TritonX-100, 5 mM EDTA, and 0.5 × sodium orthovanadate. A goat anti-FLAG tag antibody (Abcam) was used for immunoprecipitation, with magnetic protein G beads used to harvest the immunocomplexes (Dynabeads, Thermo). This was then eluted in the presence of NuPAGE LDS buffer (Invitrogen), run on a 4–12% Bis–Tris gel (Invitrogen), and transferred to a nitrocellulose membrane (iBlot, invitrogen). Mouse anti-Myc (Invitrogen) and mouse anti-FLAG (Sigma) antibodies were used to blot for the recombinant proteins.