Nupage bis tris 10 sds page gel

HEK293T cells were cotransfected with Flag-tagged target protein, HA-tagged source protein and GFP-tagged peptide or GFP. Cells were lysed 48 h after transfections with radioimmune precipitation assay buffer (50 mM Tris-HCl pH7.4, 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 10 mM Na3VO4, 10 mM sodium pyrophosphate, 25 mM NaF, 1× protease inhibitor mixture (Sigma) for 30 min at 4 °C and coimmunoprecipitated with Flag beads (Clontech). The resulting immunocomplexes were analyzed by Western blot using the antibodies indicated in Figure 3. Protein samples were separated on a NuPage Bis·Tris 10% SDS/PAGE gel (Invitrogen) and transferred to PVDF membranes. Transferred samples were immunoblotted with primary anti-HA antibodies (1:2000), followed by incubation with horseradish peroxidase-conjugated anti-rabbit secondary antibodies (Cell Signaling) (1:2500) and detected using enhanced chemiluminescence (GE Healthcare).

In order to analyze the dynamic of AKT (S473) phosphorylation, we starved RWP1 and HEK293T cells for 24 hours. We induced AKT phosphorylation by incubating cells with 10% serum (FBS) for 20 minutes in the presence or absence of peptide expression. Protein samples were separated on a NuPage Bis·Tris 10% SDS/PAGE gel (Invitrogen) and transferred to PVDF membranes. Transferred samples were immunoblotted with primary anti-AKT (1:1000) or anti-phospho-AKT (S473) (1:1000) antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (1:2500) (Cell Signaling) and detected using enhanced chemiluminescence (GE Healthcare). Stimulation of cells with serum led to an increase in AKT phosphorylation in both cell lines. Expression of KFFCTIL peptide inhibited the increase in AKT phosphorylation in RWP1 cells but not in HEK293T cells (Supplementary Fig. 10).