Un scan it

A gel of 10 cm column height with 4 mL of 15% total acrylamide monomer resolving solution was allowed to polymerize overnight with 4 μL of TEMED and 12 μL of 10% (w/v) ammonium persulfate and was cast in a Mini Prep column with a 7 mm internal diameter (Bio-Rad). Above the polymerized resolving gel, 1 mL of 5% total acrylamide monomer stacking gel was cast. An aliquot of 1 mg of purified decorin glycosaminoglycan was loaded in a solution of 10 μg/mL (w/v) Phenol Red and 25% (w/v) sucrose. Electrophoresis was performed for 8 h at a constant power of 1 W with a peristaltic pump (Econo pump, Bio-Rad) set to 0.08 mL/min and fraction collector (Model 2110, Bio-Rad) set to 3 min in conjunction-accumulated separating fractions from the Mini Prep cell (Bio-Rad). Buffer salts from electrophoresis for each fraction were removed by a strong anion exchange column (High-Capacity Mini-Q, Satorius) and thoroughly desalted by a 3 kDa spin column (Millipore) washed with LC-grade water. The extent of separation was visualized by 15% total acrylamide monomer solution using native mini-slab PAGE stained with Alcian Blue, and the molecular weight of the fractionated GAG was estimated by PAGE densitometry against heparin ladder using UN-SCANIT (Silk Scientific).

One-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed essentially as described by Laemmli et al. [31 (link)]. Protein samples were mixed with sample dilution buffer. Gels (10%) were typically run overnight at room temperature at approximately 45 V in a Hoefer SE 600 electrophoresis unit. Protein samples were electro-transferred from SDS-PAGE gels to nitrocellulose membranes in transfer buffer (25 mM Tris-base, 192 mM glycine, 20% methanol) at 200 mA for 2 h at 4 °C. Western blotting was accomplished as previously described [32 (link)]. Enhanced chemiluminescence (ECL) was used according to manufacturer’s directions (GE Healthcare, #RPN2106 or RPN2232). Santa Cruz antibodies were used for analysis of HMGCR (sc-271595) and Na⁺K⁺ ATPase (sc-28800). The GAPDH antibody was from Cell Signaling (D16H11). Band intensity was quantified using Un-Scan-It (Silk Scientific, Inc.) in the linear range of the film.

The most accurate method of determining the amount of filamentous actin (F-actin) content versus free globular-actin (G-actin) content in a cell population is to use quantitation of F-actin and G-actin cellular fractions (McCarthy et al., 2018 (link)). Vascular smooth muscle cells were treated with non-formylated peptides (control) or F-MIT (20 min, 10 μM) in the presence or absence of FPR-1 antagonist (cyclosporine H: CsH, 1 μmol/L and WRW4, 10 μmol/L) and processed according to a G actin: F actin in vivo assay (Cytoskeleton, USA). Briefly, VSMCs were lysed in a detergent-based buffer that stabilizes the G and F forms of actin. Ultracentrifugation was then used to separate them – pelleting the F actin and leaving the G actin in the supernatant. After collecting the G actin, the F actin was depolymerized. Finally, the F and G actin samples were loaded into polyacrylamide gels (10%), separated by SDS-PAGE, and transferred to PVDF membranes, as described above for Western blotting. Known quantities of actin control protein were included as standards. Densitometric analysis was performed by Un-Scan-It (Silk Scientific).