Bortezomib modulates C/EBPβ but not C/EBPα. (A) Western blot showing C/EBPα and C/EBPβ protein levels after treatment with bortezomib in the EBV(−) gastric carcinoma cell line AGS, the EBV⁺ gastric carcinoma cell line SNU-719, and the Burkitt lymphoma cell lines Akata and Rael. Western blots were also probed for β-actin as a loading control. All cells were treated with 20nM bortezomib. After 24 hours of bortezomib treatment, cells were harvested and nuclear protein extracts were prepared. (B) CAT assay performed in EBV⁺ gastric carcinoma SNU-719 cells to assess effects of C/EBP–binding sites on activation of Zp by bortezomib. Cells were transfected with a plasmid expressing Zp-CAT with wild-type C/EBP–binding sites, one C/EBP–binding site mutated, or both sites mutated. Cells were treated with 20nM bortezomib (BZ) or DMSO for 24 hours. Assays were repeated 3 times. Error bars indicate SEM. (C) ChIP assay performed in Akata cells after treatment with 20nM bortezomib for 24 hours to show ZTA promoter (Zp) DNA bound to C/EBPβ. Immunoprecipitated Zp DNA was measured by standard PCR (top panel) and analyzed on a 2% agarose gel and by real-time qPCR (bottom panel). (D) Luciferase assay performed in SNU-719 cells to assess the effects of C/EBPβ isoforms on the Zp-Luc reporter. Cells were transfected with a plasmid expressing all 3 C/EBPβ isoforms (C/EBPβ) or the indicated C/EBPβ isoforms (LAP and LIP). Transfection with an empty parent vector (vector) was used as an internal control. Assays were repeated 3 times. Error bars indicate SEM.