Figure 3.

Analysis of TFMs in the CtsZ promoter reveals a unique NFκB motif and elevated nuclear p65 levels in CtsB^−/−S^−/− RT2 macrophages. (A) Venn diagram demonstrating the overlap of predicted TFMs present in the CtsZ promoter compared with the CtsB, CtsC, CtsH, CtsL, and CtsS promoters. There are nine TFMs present in the CtsZ promoter that were not identified in any of these other cathepsin family members. Meanwhile, 83 TFMs were present in at least one of the other cathepsins but absent in the CtsZ promoter (see Supplemental Table 1 for a full list of TFMs). (B) The CtsZ promoter is shown with exons and the 5′ untranslated region (UTR) on the top bar and nine TFMs listed below. An NFκB consensus site is highlighted in red upstream of the 5′ UTR. (C) Wild-type RT2 or CtsB^−/−S^−/− RT2 BMDMs were subjected to subcellular fractionation, and lysates of the nuclear fraction (top two panels) or cytoplasmic fraction (bottom two panels) were isolated for immunoblotting of the NFκB subunit p65 and lamin A/C or p65 and actin. Results are representative of n = 3 independent biological replicates. (D) Quantification of p65—normalized to lamin A/C or actin for the nuclear and cytoplasmic fractions, respectively, using ImageJ software—showed a significant increase in p65 expression in the nucleus of CtsB^−/−S^−/− RT2 BMDMs. n = 3 replicate experiments. Statistical significance was calculated by unpaired two-tailed Student's t-test. (*) P < 0.05; (**) P < 0.01.