Antibody for β actin

Brazilian green propolis ethanolic extract (Lot. No 120928, BGP), standardized to contain 8.0% artepillin C and 0.14% culifolin was provided by Yamada Bee Company, Inc, Japan. Silica gel (N-60) and Sephadex LH-20 were from GE Healthcare (Tokyo, Japan). Precoated TLC plates (0.25 mm, silica gel Kieselgel 60F254 and silica gel 60 RP-18 F254S) were from Nacalai Tesque Inc. (Kyoto, Japan). RNAiso Plus was from Takara Bio Inc. (Kyoto, Japan). Antibodies for ERK (K-23, sc-94), phospho-ERK (E-4, sc-7383) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibody for β-actin (#4697) was from Cell Signaling Technology Japan (Tokyo, Japan). Goat anti-rabbit IgG (H+L)-HRP conjugate (#170-6515) and Immuno-Star goat anti-mouse HRP conjugate (#170-5047) were from Bio-Rad (Richmond, CA, USA). All other chemicals were of analytical grade.

Western blots were performed in according to standard procedures. HUVECs were seeded in 6-well plates and lysed with 100 μl of a cell lysis solution containing 2 μl of phenylmethanesulfonyl fluoride buffer. Equal amounts of protein (40 μg) were loaded onto 12% SDS–PAGE gels (Millipore). Primary antibodies for Casp7 (1:1000 dilution; Cell Signaling), Casp3 (1:1000 dilution; Cell Signaling), Apaf1 (1:1000 dilution; Cell Signaling) and PTEN (1:1000 dilution; Cell Signaling) were used. The antibody for β-actin (1:1000 dilution; Cell Signaling) was used as the endogenous control. The gels were run under the same experimental conditions and the original whole gel blots were included in the supplementary data. The levels of TNF-α, Apaf-1 and Casp7 protein in the serum samples obtained from the patients with CAD were measured by Enzyme-Linked Immunosorbent Assay Kits (Elabscience).

Total protein extracts from pOBs, gel electrophoresis, transfer, and visualization were performed using standard Western blotting techniques. Briefly, pOBs cells were homogenized in homemade radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor (Sigma-Aldrich) and phosphatase inhibitors (1 mM NaF, 2.5 mM, Na₄P₂O₇, 1 mM Na₃VO₄, 1 mM β-Glycerophosphate). Protein concentration was assessed using DC Protein Assay Kit II (Bio-Rad). Twenty-five micrograms (25 μg) of protein extracts were separated on a 10% SDS-PAGE, transferred to a nitrocellulose membrane, and probed with an antibody for p-DRP1[Ser616] (Cell Signaling), total DRP1 (BD Bioscence—final concentration 0.25 μg/mL) both diluted 1:1000 in 5% BSA in 20 mM Tris-buffered saline, pH 7.6, 0.1% Tween 20 (TBST), and with an antibody for β-actin (Cell Signaling) diluted 1:1000 in 5% BSA in TBST, washed, and probed with a secondary antibody conjugated with horseradish peroxidase (Bio-Rad) diluted 1:3000 in TBST, and developed using the Immobilon™ Western kit (Millipore, Watford, UK). Images were captured using the ChemiDoc™ MP Imaging System equipped with Image Lab™ Software (Bio-Rad). Band intensity analysis was performed using ImageJ Software.

Collagen-1 was purchased from either BD Biosciences (Bedford, MA) or Invitrogen (Carlsbad,CA). E-cadherin antibody was obtained from BD Biosciences. p115RhoGEF, LARG and PRG antibodies were purchased from Santa Cruz (Santa Cruz, CA). Antibody for β-actin was purchased from Cell Signaling (Danvers, MA). Monoclonal anti-vimentin antibody (clone V9) was purchased from Sigma-Aldrich, ZO-1 antibody was purchased from Invitrogen (Carlsbad,CA), hDia1(human) antibody was purchased from Proteintech (Chicago, IL). Lipofectamine 2000 and RNAiMAX was purchased from Invitrogen. siRNA oligonucleotides (p115RhoGEF: CATACCATCTCTACCGACG, LARG: GAAACTCGTCGCATCTTCC and PRG: ACTGAAGTCTCGGCCAGCT) [41] (link) were synthesized by Dharmacon. ON-TARGETplus Non-targeting siRNA was purchased from Dharmacon. siGENOME SMARTpool siRNAs library to target RhoA effector genes [27] (link) and individual siRNAs to target DIAPH1: DIAPH1 oligo#1 GAAGUGAACUGAUGCGUUU and oligo #2 GAUAUGAGAGUGCAACUAA were purchased from Dharmacon. pQCXIB-CMV/TO-DEST (w320) created by Dr Eric Campeau and pBabe-puro-Rho biosensor (created by Klaus Hahn) were obtained from Addgene. myc-DEST was a gift from Dr Adi Dubash.