HDAC6 inhibition does not influence CD20 mRNA levels and MS4A1 promoter activity. (A) cDNA prepared from mRNA of Raji cells preincubated for 24 hours with 10 µM tubacin was used for qRT-PCR amplification of CD20 and β2M with corresponding probes labeled with FAM and DABCYL. (B) cDNA from Raji cells stably transduced with either pLKO.1 or pLKO.1-shHDAC6 was used for qRT-PCR using SYBRGreen. The experiments were repeated independently 3 times. (C) Relative luciferase activity was measured in lysates from Raji cells cotransfected with either empty vector or pGL4-wild-type CD20 promoter plus pRL-TK and incubated with 10 µM tubacin for a subsequent 24 hours. Statistical significance was determined with a Kruskal-Wallis test. The experiments were repeated independently 4 times. (D) A diagram illustrating plasmids used for modification of Raji cells. (E) Raji cells stably transduced with pLVX-CD20-HISTAG were incubated with 10 µM tubacin for 48 hours. The levels of endogenous CD20 and CD20-HISTAG were assessed with western blotting with anti-CD20 monoclonal antibody. (F) Raji cells stably transduced with pLVX-CD20-FLAG were incubated with 10 µM tubacin for 48 hours. The level of CD20-FLAG was assessed with western blotting with anti-FLAG antibody. The level of CD20 (both endogenous and exogenous) was assessed with anti-CD20 monoclonal antibody. β-actin level was used as loading control. The experiments were repeated independently twice.