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. 2014 Oct 20;5(18):8765–8777. doi: 10.18632/oncotarget.2364

Figure 1. Interaction between IL-32α and BCL6 is mediated by PMA.

Figure 1

(A and B) HEK293 cells were cotransfected with a Myc-tagged–IL-32α expression vector and a FLAG-tagged-BCL6 expression vector. Twenty-four hours after transfection, the cells were pretreated for 2 h with 10 μM pan-PKC inhibitor, Gö6850 (6850), and then treated with PMA (50 nM) for an additional 3 h. Immunoprecipitation was carried out with 1 μg of myc tag antibody (A) or 2 μg of flag tag antibody (B) and 0.7 mg of whole cell lysate (WCL). Following transfection, IL-32α and BCL6 expression levels were assessed by western blot with 20 μg of WCL. (C) THP-1 EV and THP-1-IL-32α cells were transfected with a FLAG-tagged–BCL6 expression vector. After overnight incubation, cells were pretreated for 2 h with 10 μM pan-PKC inhibitor, Gö6850 (6850), and then treated with PMA (10 nM) for an additional 3 h. THP-1 cells lysates were prepared in the same way. Immunoprecipitation was carried out with 1 μg of myc tag antibody and 1 mg of WCL. (D) THP-1-IL-32α cells were co-transfected with a FLAG-tagged-BCL6 expression vector and 100 nM PKCε siRNA or nontargeting (NT) siRNA. These cells were treated with PMA (10 nM) for a additional 3 h. Immunoprecipitation was carried out in the same way, by using 1 μg of myc tag antibody. Precipitated BCL6 was detected with a flag tag antibody. IP, immunoprecipitation; WCL, whole cell lysate.