Ab23747

After co-cultured with ADSCs or BMSCs for 0, 7, 14 and 21 d in vitro, or injection of ADSCs into AAA model for 28 d, the AAA aortic wall was washed off with PBS, cut into pieces, lysed with RIPA buffer and determined protein concentration by the Bradford method. Equal amounts of protein (40 µg) were used for Western blot analysis with rabbit polyclonal antibodies to anti-elastin (abcam, ab23747, 1∶1000 dilution). Specific antibody binding was detected by horseradish peroxidase-conjugated goat anti-rabbit antibodies and visualized with ECL reagent (Santa cruz) according to the manufacturer's protocol. Antibody to β-actin was used to evaluate protein loading in each lane.

Proteins from cell lysates were separated by electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% skim milk for 2 h at RT, and then incubated with anti-elastin (1: 2000, ab23747, Abcam, Cambridge, UK), anti-SMA (1:750, ab5694, Abcam, Cambridge, UK), anti-vimentin (1:2000, ab92547, Abcam, Cambridge, UK), E-cadherin (1: 2000, 3195, Cell signalling technology, USA) and anti-β-actin (1:10,000, ab8226, Abcam, Cambridge, UK) at 4 °C overnight. Blots were incubated with anti-rabbit or anti-mouse IgG HRP conjugated antibodies (R&D System, MN, USA) at RT for 2 h after TBS-Tween-20 wash (3 times, 10 min). Substrate (SuperSignal™ West Femto Maximum sensitivity substrate, Thermo Fisher Scientific, MA, USA) was added to the membrane and images of immunoblots were captured using a ChemiDoc MP System (Bio-Rad, Hercules, USA). Some blots were cut based on molecular weight and some blots were stripped only once for housekeeping protein. Densitometry analysis was performed relative to the housekeeping protein β-actin using ImageJ (NIH, Bethesda, USA) as previously described [23 (link), 24 ].

Anti-Neuroserpin (Santacruz SC32947 and SC48360), anti-Plasmin (Santacruz 15036) and anti-methionine sulfoxide (Novus Biologicals NBPI06707SS) antibodies were used for probing western blots and immunohistochemistry of retinal sections. β-actin (Sigma, AC-40), anti-collagen (Abcam ab6586), anti-elastin (Abcam Ab23747) and anti-laminin (Abcam ab11575) antibodies were used for WB. Plasmin was from Sigma, Missouri, USA and recombinant neuroserpin protein from abcam. Plasmin enzyme activity was measured using 96-well microplate format kits from AnaSpec (Sensolyte Assay kit, AnaSpec, Inc., CA AS-72125). Fluorescent polystyrene microbeads were obtained from Invitrogen (FluoSpheres; Invitrogen, Carlsbad, CA) and dimethyl pimelimidate (DMP) was from Sigma, St. Louis, USA. Tissue type Plasminogen activator (tPA) (ab108905) and urokinase type Plasminogen activator (uPA) (ab108915) activity assay kits were obtained from Abcam. All other analytical grade reagents were from Sigma, USA.