Total p38

Lysates were centrifuged, supernatants were collected, and protein concentration was determined using a DC Protein Assay (Bio-Rad Laboratories, Hercules, CA). Samples were electrophoresed using 4–15% gradient polyacrylamide gels (Bio-Rad) and then transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked, rinsed and incubated with primary antibodies against unprenylated Rap1A (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), p-P38, total P38, p-Pak1, total Pak1 (Cell Signaling Technology, Inc., Danvers, MA), cleaved PARP-1 (Cell Signaling) and cleaved caspase-3 (eBioscience). After overnight incubation at 4°C, membranes were washed and incubated with their corresponding secondary antibody conjugated with horseradish peroxidase (HRP). Protein bands were detected with an enhanced chemiluminescence detection kit (GE Healthcare, Piscataway, NJ). β-Actin (Sigma Aldrich) and vinculin (Santa Cruz) were used as loading controls.

Expression of activated pro-inflammatory (p38 and JNK) and anti-inflammatory proteins (ERK, SMAD) was determined using our standard Western blot technique (17 (link)). In brief, arterial tissue was collected, preserved in RNAlater, and stored at −80^oC until homogenization. Post homogenization, proteins were estimated using Bradford Assay. Proteins were then analyzed using 10% SDS-PAGE gel electrophoresis and transferred onto a PVDF membrane (Bio-Rad) using a Bio-Rad Semi-Dry Transfer Cell at 15V for 45 minutes. Membranes were blocked for 1 hour at room temperature with 5% milk, decanted, and incubated overnight with the appropriate primary antibody in 5% milk: p- JNK (Cell Signaling, 1:1000, #4668S), Total JNK (Cell Signaling, 1:1000, #9252S), p- p38 (Cell Signaling, 1:1000, #4631S), Total p38 (Cell Signaling, 1:1000, #9212S), p- ERK (Santa Cruz, 1:1000, sc7383), β-actin (1:1000, Cell Signaling, #4967L) or p-SMAD (Cell Signaling, 1:1000, #13820S). After overnight incubation, membranes were washed and incubated with HRP-conjugated anti-rabbit IgG (Cell Signaling, 1:1000, #7074S) or anti-mouse IgG (Santa Cruz, 1:1000, sc2064) for one hour at room temperature. Membranes were washed and incubated with ECL (Thermo Scientific, #32106). Densitometry analysis of protein expression was completed using Image J as previously reported (9 (link)).