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. 2004 Feb 26;23(5):1112–1122. doi: 10.1038/sj.emboj.7600125

Figure 7.

Figure 7

SPAK is required for PKCθ-A/E-induced AP-1, but not NF-κB, activation. (A, B) Jurkat E6.1 cells were transfected with empty vector (left lane) or c-Myc–PKCθ-A/E (four right lanes) plus the indicated pSuper, pSuper–RNAi-1 or pSuper–RNAi-2 vectors, β-Gal reporter plasmid and AP-1 (B; upper panel) or NF-κB (B; lower panel) reporter genes. (A) Expression levels of SPAK mRNA (measured by real-time RT–PCR and expressed as arbitrary units), and SPAK or PKCθ protein (measured by immunoblotting with anti-SPAK or anti-c-Myc antibodies, respectively). (B) Normalized AP-1 (upper panel) or NF-κB (lower panel) activities in the same cells. (C) Jurkat-TAg cells were cotransfected with the indicated combinations of empty vector, c-Myc–PKCθ-A/E and/or different SPAK constructs together with AP-1–Luc and β-Gal reporter genes. Cell extracts were prepared 24 h later, and normalized AP-1 activity (upper panel) or protein expression levels (two lower panels) were determined. Mean values of duplicate determination±s.e. are shown. Similar results were obtained in two additional experiments.