Vascular smooth muscle cells were treated with non-formylated peptides or F-MIT (20 min, 10 μM). After treatment, cells were washed with an ice-cold phosphate-buffered saline (PBS) solution. Complete Lysis-M, including phosphatase inhibitor cocktail (PhosSTOP) (both Roche, Indianapolis, IN, USA), was then applied to each plate and allowed to remain on ice for 30 min. Cells were then harvested. Protein concentration was first determined and then equal quantities of protein were loaded into polyacrylamide gels (8–12%) and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Gels were then transferred to polyvinylidene difluoride (PVDF) membranes (Thermofisher). The membranes were blocked with 5 % nonfat dry milk and incubated overnight at 4°C with primary antibodies raised against anti-RhoA (1:2000; Cell Signaling), ROCK (1:1000; Cell signaling), L-type calcium channel (Cav 1.2, 1:500, abcam), PKCα (1:1000, BD Bioscience), AHNAK (1:1000, Thermofisher), pMYPT1Thr696 (1:1000, Cell Signaling), MYPT1 (1;1000, BD Transduction). β-actin (1: 40:000, Sigma) was used as the loading control. Densitometric analysis was performed by Un-Scan-It (Version 6.1) (Silk Scientific, USA).
Un scan it version 6.1
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3 protocols using un scan it version 6.1
1
Signaling Mechanisms in Vascular Smooth Muscle
2
Western Blot Protein Quantification
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We ran SDS-PAGE gels (gradient 4–20 %) in HEPES-SDS (NuSep Inc. North America). Volumes of samples were loaded with the intent of standardizing protein mass to facilitate quantitation. Actin was used as a loading control when possible. Proteins were transferred to PDVF membranes via electroblotting using Bio-Rad’s vertical system. Membranes were stained for total protein with AMRESCO ProAct Membrane Stain and erased before proceeding to immunodetection by the indirect method. The main supplier of primary antibodies in both rabbit and mouse hosts was Abcam (Cambridge, MA, USA). There was considerable variation in the “titer” of the antibodies. Therefore, it was not possible to obtain data on the relative amounts of each type of SOD from the Western blots (such estimates were made from the relative enzyme activity as described below). The secondary antibodies (LI-COR® Biosciences) were detected and scanned with the Odyssey Infrared Imaging System at 680 nm (red) and 800 nm (green). Band intensity was quantified using UN-SCAN-IT Version 6.1 (Silk Scientific). Differences in band density among treatments were examined using 1-way ANOVA (α = 0.05).
3
Western Blot Analysis of Tight Junction Proteins
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Western blotting of TJ protein expressions was performed as described previously (33) . The primary antibodies against occludin and ZO-1 were purchased from Abcam. Relative changes in protein expression were estimated from the pixel density using UN-SCAN-IT version 6.1 (Silk Scientific Inc.), normalised to b-actin and calculated as target protein expression:b-actin expression ratios.
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