Pcdna flag hdac4

Myr-Akt plasmids were kindly provided by Professor Chien-Huang Lin (Taipei Medical University, Taipei, Taiwan). PcDNA-FLAG-HDAC1 (plasmid 13820), pcDNA-FLAG-HDAC4 (plasmid 13821), pcDNA-FLAG-HDAC6 (plasmid 13823) were purchased from Addgene (Cambridge, MA, http://www.addgene.org). Transfections for BJAB cells were done by nucleofection using nucleofector solution L, program T-016, according to the manufacturer's instructions (Amaxa, Inc.). A total of 4×10⁶ cells were used per transfection and mixed with 0.5 or 1 μg of myr-Akt, pcHDAC1, pcHDAC4 and pcHDAC6. After transfection, BJAB cells were recovered for 24 h incubating at 37°C.

Cells were seeded into 6-wells for overnight and then transfected with HDAC1, HDAC4, HDAC6, or MEK plasmids and ERK siRNA by using Lipofectamine 2000 for 8 h. PcDNA-FLAG-HDAC1 (plasmid 13820), pcDNA-FLAG-HDAC4 (plasmid 13821), pcDNA-FLAG-HDAC6 (plasmid 13823), and GFP-MEK (plasmid 14746) were purchased from Addgene (Cambridge, MA, USA), and ERK siRNA (s11137) was purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). Refreshed culture medium for overnight and then treated with MPT0E028, PD98059, or both drugs for 48 h. Cell lysates were collected for western blot analysis.