NFAT facilitates TMP21 gene expression at both the mRNA and protein level. HEK293 (A) and SH-SY5Y (B) cells were transfected with NFAT expression plasmid or NFAT siRNA (sc-29412, Santa Cruz Biotechnology, Inc.). Total RNA was extracted. Semi quantitative RT-PCR was performed to detect the mRNA levels of TMP21 and β-actin. Specific TMP21 and β-actin coding sequence primers were used to amplify the TMP21 and β-actin cDNA, as described in Materials and Methods. The RT-PCR products were analyzed on 1% agarose gels. β-actin was used as internal control. (C) The ratio of TMP21 to β-Actin mRNA was quantitated by Kodak Image Analysis. Shown is the Mean+S.E.M., and n = 4. *p < 0.001 relative to controls by ANOVA with post-hoc Newmann-Keuls test. (D and E) Western blot assay was then performed to analyze cell lysates from NFAT-transfected cells and NFAT siRNA on 16% Tris-Glycine gel. TMP21 protein was detected using rabbit anti-mouse anti-TMP21 ployclonal antibody T21 and β-actin was used as an internal protein control. Overexpression of NFAT increased TMP21 protein generation and NFAT siRNA decreased the protein levels in HEK293 (D) and SH-SY5Y (E) cells. (F) Quantitative analysis of the generation of TMP21. Values are Means ± S.E.M. and n = 4. The protein levels are expressed as a percentage of the levels in control cells. * p < 0.001 relative to controls by ANOVA with post-hoc Newmann-Keuls test.