Immuno (Dot) blot
To determine whether the DNA binding proteins were related to NFκB and NFAT, dot blots using a SRC96D SNS minifold I dot blotter (Schleicher & Schüll, Dassel, Germany) were performed with the following positive controls and antibodies (all from Santa Cruz Biotechnology, Heidelberg, Germany): positive controls (10 ng/dot): NFκB p52 (80 kD) sc-4095WB, c-Rel (61 kD) sc-4030WB; Jurkat nuclear extract, PMA-stimulated; primary antibodies (1 μg/dot): anti-NFκB p50 (NLS) sc-114, anti-NFκB p65 (A) sc-109, anti-NFκB p52 (447) sc-848, a rabbit polyclonal IgG1 antiserum, anti-c-rel (N466) sc-272, anti-NFATc2 (M-20) sc-1151, anti-NFATc1 (K-18) sc-1149, two goat polyclonal IgG1 antisera; secondary antibodies (0.8 μg/dot): goat anti-rabbit IgG AP-conjugate sc-2007, and donkey anti-goat IgG AP-conjugate sc-2022. The nuclear proteins were blotted onto a nitrocellulose membrane (Bio-Rad). After blocking with PBS/3% BSA (Fluka, Deisenhofen, Germany), the blot was incubated overnight with the primary antibodies diluted 1:2000 in PBS/1% BSA, washed again and incubated with the alkaline phosphatase-conjugated secondary antibody (1:500 in PBS/1% BSA). The blot was developed using the Vectastain® staining kit (Vector Laboratories Inc. Burlingame, CA, USA).