Gelatin gel

Protein from ASC tissues and SMC was extracted and the concentration was determined as described. To measure MMP‐2 activity from tissue, 5 μg of total protein was resolved by nondenaturing electrophoresis through a 10% gelatin gel (Invitrogen, Carlsbad, CA) according to manufacturer's instructions. Experiments included 7 to 10 mice per group (WT male=10, WT female=10; Fbn1 male=10; Fbn1 female=10; Fbn1 male flutamide treated=7; Fbn1 male vehicle control treated=7). For in vitro studies, Fbn1^C1039G/+ SMC (2.0×10⁵ cells/mL) were cultured to confluence in SMC media and starved with serum‐free medium for 24 hours, then treated with hormones for 24 and 48 hours. Following drug treatment, SMC culture supernatants were collected and concentrated using Amicon Ultra‐4 centrifugal Filters (Ultracel‐30K) (Millipore sigma, Billerica, MA). Two μg of the concentrated protein was resolved by nondenaturing electrophoresis through a 10% gelatin gel (Invitrogen). The molecular sizes of gelatinolytic activities were determined using 10 μL of MMP standard marker (Cosmobio, Tokyo, Japan). Quantification by densitometry was performed using ImageJ software (National Institutes of Health) on inverted images of gel. Experiments included n=6 per group.