Ab125025

Immunohistochemistry detection of Rho A, ROCK1, ROCK2, GLUT1, HK2, PDK1, PFK1, LDHA, OPN and RUNX2 in human aortic valve leaflets was performed. 4% PFA-fixed paraffin sections (5μm thick) were deparaffinized using dewaxed solution and hydrated using decreasing concentration of ethyl alcohol (100%, 90%, 80%, 70%, 60%), and then incubated at 65 °C for 20 min in antigen unmasking solution using PT Module-Lab Vision (Thermo Fisher Scientific) for antigenic retrieval. Following several washes (3 x 5 min) in PBS, paraffin sections were blocked for 30 min using 5% bovine serum albumin (BSA). After blocking, primary antibodies against Rho A (abcam, ab54835), ROCK1 (abcam, ab97592), ROCK2 (abcam, ab125025), GLUT1 (abcam, ab115730), HK2(abcam, ab209847), PDK1 (abcam, ab202468), PFK1 (CST, #8164), LDHA (abcam, ab52488), OPN (abcam, ab63856) and RUNX2 (abcam, ab192256) were diluted at optimized concentrations in 1% BSA and incubated with the sections in a wet box overnight at 4 °C. Subsequently, appropriate secondary antibodies were applied for 30 min at room temperature. Following secondary antibody incubation, the positive staining was detected using an UltraSensitive SP IHC Kit and DAB Kit, and hematoxylin staining was used for nuclear counterstaining. Images were visualized using a microscope (CKX53, Olympus), and Image J software was applied for data quantification.

After 48 h of transfection, cells were harvested, and proteins were extracted. Then, a BCA protein assay kit was used to quantify the protein concentration, and proteins were separated on 10% (FAK and ROCK2) or 12% (RHOA) SDS-PAGE gels and transferred to PVDF membranes (Millipore, USA). Then, the membranes were blocked with 5% skim milk at 37 °C for one and a half hours. The primary antibodies were as follows: anti-ROCK2 (ab125025, Abcam, USA), anti-FAK (ab40794, Abcam, USA), anti-RHOA (ab187027, Abcam, USA) and anti-β-ACTIN (ab8227, Abcam, USA).

Proteins that were extracted from ox-LDL-treated cells were separated with Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (cat. no. P1200; SDS-PAGE, Solarbio) and polyvinylidene difluoride (PVDF) membrane (Amersham, Munich, Germany), and then PVDF membrane was blocked with TBST buffer containing 5% bovine serum albumin. The protein bands were incubated with primary antibodies: CyclinD1 (ab16663, 1:1000, Abcam, Cambridge, MA, USA), cleaved-caspase 3 (ab2302, 1:1000, Abcam), ROCK2 (ab125025, 1:1000, Abcam), GAPDH (ab9485, 1:1000, Abcam), and the secondary antibody (1:5000, ab205718, Abcam), respectively. Finally, the protein bands were incubated with ECL Western Blotting Substrate (cat. no. PE0010; Solarbio) and observed by a gel imaging analysis system. The original western blots were showed in Additional file 1.

Cells were lysed in RIPA buffer supplemented with protease inhibitor cocktail (Sigma-Aldrich). Conditioned media were mixed with four volumes of chilled acetone. Proteins were precipitated overnight at −30 °C and dissolved in RIPA buffer. Protein concentrations were measured by bicinchoninic acid protein assay (Thermo Scientific Inc., Waltham, MA, USA). Cell lysates were boiled in sodium dodecyl sulfate loading dye. Equal amounts of total protein were separated on 4–20% mini-Protean TGX gradient gels (Bio-Rad) and transferred to polyvinylidene difluoride membranes (Immobilon P). The membranes were incubated with the primary and secondary antibodies. Finally, bands were visualized with the electrochemiluminescence reagents (GE Healthcare, Chicago, IL, USA) and using an ImageQuant LAS-4000 mini system (GE Healthcare). The antibodies for immunoblotting were as follows: anti-β-catenin (1:1000, 610153, BD Biosciences), anti-SLPI (1:1000, AF1735, R&D Systems), anti-ROCK1 (1:1000, ab134181, Abcam, Cambridge, UK), anti-ROCK2 (1:1000, ab125025, Abcam) and anti-β-actin-HRP (1:5000, ab20272, Abcam), goat anti-mouse IgG-HRP (1:1000, sc-2005, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-goat IgG-HRP (1:1000, sc-2768, Santa Cruz Biotechnology).

The antibodies used were as follows: rabbit anti-RhoA (1 : 300, Abcam, ab187027), rabbit anti-ROCK (1 : 200, Abcam, ab125025), rabbit anti-CD 68 (1 : 200, Abcam, ab125212), rabbit anti-CD80 (1 : 200, Abcam, ab215166), rabbit anti-CD163 (1 : 200, Abcam, ab182422), rabbit anti-CD206 (1 : 200, Abcam, ab64693), rabbit anti-inducible nitric oxide synthase (iNOS, 1 : 200, Abcam, ab178945), mouse antitumor necrosis factor-α (TNF-α, 1 : 200, Santa Cruz, sc-52746), mouse anti-interleukin-6 (IL-6, 1 : 300, Abcam, ab9324), and mouse anti-IL-10 (1 : 200, Santa Cruz, sc-365858).