Iκb sc 371

Lysate homogenates of renal tissues or cells were prepared, and Western blotting was performed, as previously described unless otherwise indicated.^{49 (link)} Each nitrocellulose membrane was incubated with antibodies to phospho–JNK (4668, 1:1000, Cell Signaling Technology, Danvers, MA), NF-κB p65 (sc-7151, 1:500, Santa Cruz Biotechnology), phospho-IκB (sc-8404, 1:500, Santa Cruz Biotechnology), IκB (sc-371, 1:500, Santa Cruz Biotechnology), IKKα/β (sc-7607, 1:500, Santa Cruz Biotechnology), phospho-IKKα/β (sc-2697, 1:500, Santa Cruz Biotechnology), laminin B (sc-6216, 1:500, Santa Cruz Biotechnology), FGF1 (Abcam, 1:2000), or glyceraldehyde 3-phosphate dehydrogenase (1:5,000, Santa Cruz Biotechnology). The immunoreactive bands were then detected by incubating with secondary antibody (sc-2004, Santa Cruz Biotechnology) conjugated with horseradish peroxidase and visualized using enhanced chemiluminescence reagents (Bio-Rad, Hercules, CA). All Western blots were repeated at least 3 times. The density of the immunoreactive bands was quantified using ImageJ software (NIH, Bethesda, MD). For densitometric quantification of JNK, the total amount of both 46 kD JNK1 and 54kD JNK2 was calculated.