Hyblot es autoradiography film

A nonradioactive chemiluminescent EMSA kit (Active Motif, Carlsbad, CA) was used to examine whether mouse Klf13 binds to the putative binding site on the mouse IL-4 proximal promoter. The oligonucleotides used as probes in gel shift assays were end-labeled at their 5′ ends with biotin, and the sequences are as follows: RANTES: forward, 5′-GCTATTTTGGAAACT CCCCTT-3′; reverse, 5′-AAGGGGAGTTTCCAAAATAGC-3′; IL-4: forward, 5′-GACACCAGCACCCTCGGACAC-3′; reverse, 5′-GTGTCCGAGGGTGCTGGTGTC-3′. Competitor oligonucleotides were the same sense and antisense sequences as above without the 5′-biotin label. The 21-bp probe and competitor DNA consisted of cDNA fragments annealed by heating to 95°C and slowly cooled to room temperature. All EMSA experiments were performed on 5% polyacrylamide gels in 0.5× Tris-borate-EDTA buffer. Each EMSA reaction mixture contained 50 ng poly(deoxyinosinic-deoxycytidylic) acid, 1× LightShift EMSA Kit binding buffer, 20 fmol biotin-labeled DNA probe and 5 μg 293T cell nuclear extract overexpressing mouse Klf13, and 4 pmol nonbiotinylated competitor DNA. EMSA gels were electroblotted onto GeneScreen Plus Hybridization Transfer Membrane (PerkinElmer). Signal development followed the LightShift Chemiluminescence EMSA Kit protocol with HyBlot ES Autoradiography Film (Denville Scientific) for luminescence detection.

Protein lysates from treated cells or aortic tissues were prepared as described previously.^{13 (link),14 (link)} Protein samples (15 μg per lane) were subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and were transferred to polyvinyl difluoride membranes. The membranes were blocked for 1 hour in blocking solution of Tris-buffered saline containing 5% nonfat dried milk and 0.5% Tween 20 and then were incubated with a primary antibody. Next, the membranes were washed and incubated with horseradish peroxidase–conjugated anti-rabbit or anti-mouse secondary antibody. Antibodies used for western blotting are listed in Table I in the Data Supplement. Protein bands were visualized by using Clarity Enhanced Chemiluminescence (Bio-Rad Laboratories, Inc., Hercules, CA) and were exposed with HyBlot ES autoradiography film (Denville Scientific Inc., Holliston, MA). The blots were quantified with densitometry by using the Quantity One imaging program (Bio-Rad). Protein levels were normalized to those of β-actin and were expressed as a percentage of the untreated control.