Anti c myc clone 9e10

Whole cell lysates were generated from transiently transfected HEK293T cells as described above. A BCA assay was used to determine the protein concentration of the lysates and equal amounts of protein were mixed with loading buffer and ran on a 4%–20% SDS-PAGE gradient gel. Proteins were transferred to a nitrocellulose membrane and subjected to western blot analysis using anti-c-Myc clone 9E10 (1:1000, Sigma) or anti-FLAG M2 (1:1000, Sigma). Anti-GAPDH (1:4000, Fisher/Meridian) was used as a loading control.

Lysates of 293FT cells transfected with the expression vectors were incubated with anti-c-myc (clone 9E10, Sigma-Aldrich, St Louis, MO, USA) or anti-Flag M2 antibodies (Sigma-Aldrich) for 1 h at 4 °C, and immune complexes were incubated with protein G-sepharose (GE Healthcare, Little Chalfont, UK) for 1 h at 4 °C. For immunoprecipitation of HBZ-binding proteins, rabbit polyclonal anti-HBZ antibody^{40 (link)} was incubated with the lysates of MT4 or splenocytes from HBZ-Tg mice. The following antibodies were used for immunoblotting: anti-Flag (M2, Sigma-Aldrich), anti-His (PM002, MBL, Nagoya, Japan), anti-Actin (AC-40, Sigma-Aldrich); anti-Rb (clone 4H1 from Cell Signaling Technology (Danvers, MA, USA) and clone EPR17512 from Santa Cruz Biotechnologies, Dallas, TX, USA), anti-E2F-1 (clone c-20, Santa Cruz Biotechnologies), anti-HDAC3 (polycolonal, Abcam, Cambridge, UK) and anti-Tax (clone MI73).^{35 (link)} Peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG were purchased from GE Healthcare. All IP and immunoblotting in this study were repeated independently at least three times, and the representative results are shown.