Western blotting and immunofluorescence

Total proteins from lysates of tissues and U-87MG and U251 cell lysates were extracted by re-suspending the cell pellets in RIPA buffer (150 mM NaCl, 50 mM Tris (pH 7.4) and 1% Triton X-100). Approximately 55 μg of total protein per sample was separated by SDS-PAGE and then transferred to a nitrocellulose membrane. Western blot analyses were performed with polyclonal antibodies against Pygo2, CyclinD1, CyclinA, C-myc, CD44 (Santa Cruz Biotechnology, USA), H3K4me3, anti-H3K4me2, and H3 (Sigma, USA), monoclonal β-actin antibody as control (Sigma, USA). Immunofluorescence was performed using a standard procedure with primary antibodies against Pygo2 (Santa Cruz Biotechnology, USA) and GFAP (Sigma, USA).