Enhanced chemiluminescence plus

Western blotting was carried out as in previous studies (e.g. refs.^{5 (link),7 (link),10 (link)}). Transfected HEK-293 cells were incubated with collagen or collagen-mimetic DDR-selective peptides in the presence or absence of anti-DDR1 mAbs, as indicated in the Figure legends. Cells were lysed in 1% Nonidet P-40, 150 mM NaCl, 50 mM Tris, pH 7.4, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 50 µg/ml aprotinin, 1 mM sodium orthovanadate, and 5 mM NaF. Aliquots of the lysates were analysed by reducing SDS-PAGE on 7.5% polyacrylamide gels. The gels were blotted onto nitrocellulose membranes. Blots were first probed with phospho-specific Abs, followed by horseradish peroxidase conjugated goat anti-rabbit secondary Abs, then stripped in Antibody Stripping Solution (Alpha Diagnostic International, San Antonio, Texas) and re-probed with anti-DDR1 Abs followed by horseradish peroxidase conjugated goat anti-rabbit secondary Abs. Signal detection was performed using Enhanced Chemiluminescence Plus (Amersham Biosciences) on a Typhoon FLA 9500 Imager (GE Healthcare Biosciences). Densitometry analysis of protein band intensities was performed using ImageStudio^TM Lite (LI-COR Biosciences, UK).

Membrane proteins were solubilized in sodium dodecyl sulfate buffer containing 5% 2-mercaptoethanol and separated by polyacrylamide gel electrophoresis as previously described [25] (link). The following antibodies were used: rabbit polyclonal anti-TRPC6 (dilution 1∶500, Sigma-Aldrich, St Louis, MO); mouse monoclonal anti-NCX1 (dilution 1∶500; R3F1; Swant, Bellinzona, Switzerland); mouse monoclonal anti-SERCA2 (sarco−/endoplasmic reticulum Ca²⁺ pump; dilution 1∶1,500; Affinity Bioreagents, Golden, CO). Gel loading was controlled with monoclonal anti-β-actin antibodies (dilution 1∶10,000; Sigma-Aldrich). After washing, membranes were incubated with anti-rabbit horseradish peroxidase-conjugated IgG for 1 h at room temperature. The immune complexes on the membranes were detected by enhanced chemiluminescence plus (Amersham Biosciences, Piscataway, NJ) and exposure to X-ray film (Eastman Kodak, Rochester, NY). Quantitative analysis of immunoblots was performed by using a Kodak DC120 digital camera and 1D Image Analysis Software (Eastman Kodak). In these studies we used aorta; the blotting of low abundance membrane proteins in arterioles <300 µm is often not feasible without pooling vessels from large numbers of animals. In general where smaller arteries have been studied simultaneously with aorta the primary changes in NCX1 are similar [28] (link), [29] (link).