Figure 6. Neutrophil-specific gene expression is inhibited by constitutively active CKLiK.

(A) Shown are the results of two northern assays that were performed using total RNAs from uninduced and ATRA-induced EPRO cells expressing the empty vector, constitutively active versions of CaMKKα (CaMKK₄₁₃, clone 2) or CKLiK (CKLiK₂₉₆, clone 9), or the kinase dead version of CKLiK₂₉₆ (CKLiK₂₉₆-KD, clone 12). The blots were sequentially probed with ³²P-labelled cDNAs for the secondary granule genes lactoferrin (LF) and neutrophil gelatinase (NG), the transcriptional regulator PU.1, and either the NADPH oxidase component gp91^phox (left panel) or the neutrophil-specific transcriptional regulator C/EBPε (right panel). A β-actin probe was also used to demonstrate amounts of RNA in each lane. (B) A northern assay (left panel) demonstrates that two independently generated lines of EPRO cells that express CKLiK₃₈₅-CA exhibit reduced lactoferrin and gp91^phox transcript expression as compared to cells expressing the empty vector. A Western assay was also performed (right panel), which demonstrates that expression of gp91^phox proteins are undetectable in EPRO-CKLiK385-CA cells whereas expression of either p47^phox or p67^phox is normally upregulated. Levels of actin expression are shown in the bottom panels of each figure.