Nb110 41568

Secreted proteins were first concentrated using Amicon Ultra Cel-10 centrifugal filters (MWCO 10.000; Millipore, Bedford, MA, USA) as previously described [94 (link)]. Cellular lysates or secreted proteins were resolved by 10% SDS-PAGE, and the proteins were electrophoretically transferred onto nitrocellulose membrane (Bio-Rad, Herwles, Hercules, CA, USA) and analyzed as previously [24 (link)]. Secreted and cellular protein bands of human melanoma cells and fibroblasts were quantified using Image Studio Lite (LI-COR Biotechnology, Lincoln, NE, USA). A rabbit polyclonal antibody recognizing the C-terminal domain of LOX (L4669; Sigma-Aldrich), a rabbit polyclonal LOX propeptide antibody to LOX-PP (NB110-41568; Novus Biologicals, Littleton, CO, USA), and a rabbit polyclonal antibody to LOXL2 (ab96233; Abcam, Cambridge, UK) were used to detect the respective proteins. Secreted protein samples were verified to contain comparable amounts of total proteins by silver staining. Mouse monoclonal antibodies to alpha-tubulin (DM1A; Abcam) and actin (JLA20; Merck, Millipore, Billerica, MA, USA) were used as loading controls for cellular proteins.

Mouse aortas were fixed overnight in 4% paraformaldehyde/0.1 M PBS (pH 7.4), embedded in paraffin, and sectioned into 5 mm sections. Deparaffinized sections were rehydrated, subjected to antigen retrieval (10 mM citrate pH 6.0) and blocked with 10% serum/PBS. Antibodies against LOX (ab31238, Abcam), LOX-PP (NB110-41568, Novus Biologicals) and MAC3 (sc-19991, Santa Cruz Biotechnology Inc., Europe) were used. Sections were incubated with the corresponding biotinylated secondary antibodies. Immunocomplexes were detected after incubation with Vectastain Elite ABC reagent (PK6100; Vector Laboratories, Burlingame, CA, USA) and 3,3′-diaminobenzidine substrate.

Whole-cell extracts were obtained from VSMC as described^{42 (link)}. In some experiments, supernatants from TgLOX or wild-type VSMC concentrated with Amicon Ultra 10 K filter units (Millipore) were used^{43 (link)}. Whole-cell extracts and concentrated cell supernatants were resolved by SDS-PAGE and transferred to 0.45 μm polyvinylidene difluoride membranes (Immobilon, Millipore). Blots were incubated with antibodies directed against LOX (ab31238, Abcam), LOX-PP (NB110-41568, Novus Biologicals) and PCNA (FL-261, Santa Cruz Biotechnology). Bound antibodies were detected after incubation with a HRP-conjugated goat anti-rabbit IgG and using the SuperSignal West Dura Extended Duration Substrate (Pierce). Equal loading of protein was verified by Ponceau staining and by β-actin (A5441, Sigma) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; MAB374, Millipore) signal as stated.