Trans blot sd semi dry electrophoretic transfer cell system

Proteins from gastrocnemius muscle were isolated in RIPA buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.05% NP-40, 1% deoxycholate, 0.1% SDS), and proteins from isolated endothelial cells and pericytes were isolated in NP40 Cell Lysis Buffer (ThermoFisher Scientific, FNN0021), both supplemented with protease inhibitor cocktail (Sigma-Aldrich, P8340) and phosphatase inhibitor cocktail Set V (Millipore, 524629). Samples were heated at 37°C for 30 minutes in SDS-PAGE sample buffer, loaded in a precast 4%–20% polyacrylamide gradient gel (Bio-Rad), and subjected to electrophoresis. Proteins were transferred onto a PVDF membrane for 90 minutes at 50 mA using the Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell System (Bio-Rad). Membranes were blocked in 5% milk for 1 hour and then incubated overnight with the primary antibodies (Supplemental Table 3). Membranes were washed and incubated with secondary antibodies for 1 hour (Supplemental Table 3). Detection was performed using SuperSignal West Femto (ThermoFisher Scientific, 34096) and visualized with a G-Box Chemi-XT4 (SynGene). GAPDH was used as reference protein.

Proteins for the western blot were extracted according to the manufacturer’s protocol with TRIzol (Invitrogen) and protein in microliters was quantified with QuantiPro BCA Assay Kit (Sigma-Aldrich). A total of 30 μg of protein was loaded per lane and resolved in 10% resolution gel at 120 V for 2 h and transferred to a nitrocellulose membrane (LI-COR) at 20 V for 70 min with the Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell system (Bio-Rad Laboratories). Transfer and quality were checked with Ponceau staining (VWR) and washed thoroughly with 1× PBS with 0.05% Tween 20 (PBST). Membranes were incubated with primary antibody (anti-p53, 1:1,000 dilution, catalog no. 55342, AnaSpec and anti-actin 1:1,000 dilution, catalog no. A2066, Sigma-Aldrich) overnight at 4 °C with gentle shaking after 1-h blocking in 5% skimmed milk (Sigma-Aldrich) in 0.05% PBST at room temperature with gentle shaking. After three washes with 0.05% PBST, membranes were incubated with secondary antibody (anti-rabbit, 1:10,000, catalog no. sc-2357, Santa Cruz Biotechnology) for 1 h at room temperature with gentle shaking. This was followed by three washes with 0.05% PBST and membranes were then revealed with Amersham ECL Select (Cytiva) using the Fusion Solo system (Vilber Lourmat).

Anti-serum against CPILE-a and the trypsin-treated rCPILE-b was prepared in rabbits at Funakoshi Co., Ltd (Tokyo, Japan). The materials, which were precipitated by 60%-ammonium sulfate saturation, from the cultures incubated strain W5052 in modified DS medium were used as a test material. A western blot analysis was performed after protein separation on SDS-PAGE gels, followed by electrophoretic transfer to Immun-Blot PVDF membranes (Bio-rad) using a Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell system (Bio-rad). The detection of the homologues was accomplished by incubating the membrane with a blocking buffer (10% Skim milk in PBS, BD) overnight at 4°C. The membrane was then incubated with 1,000-fold diluted polyclonal antiserum in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) for 1 hr. After being washed, the membrane was treated with goat anti-rabbit IgG coupled with horseradish peroxidase (Pierce) in TBST for 1 hour. The reaction was visualized by the use of a Metal Enhanced DAB Substrate Kit (Pierce) according to the manufacturer's instructions.