Polyvinylidene fluoride microporous membrane

The heart tissues were quickly collected and frozen in liquid nitrogen for further research. Then, heart tissues were lysed in ice-cold RIPA buffer with a 1% protease and phosphatase inhibitor cocktail. The protein concentration in the lysis buffer was determined by bicinchoninic acid (BCA) protein assay reagent kit (Thermo Scientific, United States), and 20 μg proteins were separated by SDS-PAGE (8% or 10%) and transferred to a polyvinylidene fluoride microporous membrane (Millipore, Burlington, MA). Subsequently, membranes were blocked with 5% skim milk or 1% BSA and incubated with specific primary antibodies for toll-like receptor 4 (TLR4) (Abcam, rabbit Ab, 1:300), interleukin receptor-associated kinase 1 (IRAK1) (Abcam, rabbit Ab, 1:1,000), tumor necrosis factor receptor-associated factor 6 (TRAF6) (Santa Cruz, mouse Ab, 1:500), nuclear factor-kappa B (NF-κB) (Cell Signaling Technology, rabbit mAb, 1:1,000), type I collagen (collagen I) (Bioss, rabbit Ab, 1:1,000), β-actin (Proteintech, mouse Ab, 1:4,000–5,000) at 4°C overnight and followed by incubation with appropriate peroxidase-conjugated secondary antibodies. ImageJ software (NIH) was used for quantitative analysis.

Protein samples for western blot analysis were prepared as described previously [22] (link). Briefly, MSCs (passages 3–5) were washed three times with ice-cold PBS and then treated with lysis buffer (50 mM Tris-HCl, pH 7.5, containing 2% SDS (Sigma-Aldrich) and a protease inhibitor cocktail (Roche, Mannheim, Germany). Samples were centrifuged for 1 h at 18,000× g at 4°C. The supernatants were collected as whole cell lysates. Protein concentrations were estimated using a DC protein assay (Bio-Rad) with a bovine serum albumin standard. Equal amounts of proteins (10 µg) were resolved by SDS-polyacrylamide gel electrophoresis on 4–20% acrylamide gradient gels (Bio-Rad) and then transferred onto a polyvinylidene fluoride microporous membrane (Millipore, Billerica, MA). The membranes were blocked with a blocking reagent (Toyobo, Tokyo, Japan) and then incubated with each primary antibody. The primary antibodies used were: rabbit anti-PCAF, rabbit anti-HIF-1α (Cell Signaling Technology), rabbit anti-VEGF (Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-β-actin (Cell Signaling Technology). After washing, the membranes were incubated with a peroxidase-labeled secondary antibody (Nichirei, Tokyo, Japan) and visualized using Immunostar LD (Wako). Images were captured digitally using a ChemiDoc XRS+ (Bio-Rad) and analyzed by Image Lab 2.0.1 software (Bio-Rad).

Proteins were separated by 10% SDS-containing polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride microporous membrane (Millipore, MA, USA). The membrane was probed with primary antibodies including HAb18, C-19 (Santa Cruz Biotechnology), anti-MMP-2 (Santa Cruz Biotechnology), anti-p-ERK1/2, anti-p-FAK, anti-p-Akt, anti-p-EGFR, anti-ERK1/2, anti-Akt, anti-EGFR (Cell Signaling Technology, Danvers, USA), anti-FAK (BD), and anti-α-tubulin antibodies (Santa Cruz Biotechnology).

Cell pellets were solubilized in electrophoresis sample buffer, sonicated for 10 s, and boiled for 10 min. The protein concentration of cell lysates was measured, and 3 mg protein was separated by SDS-PAGE, and then transferred to a polyvinylidene fluoride microporous membrane (MILLI-PORE, Billerica, MA). Membranes were blocked in PBS with 5% milk powder and probed with anti-AKT (ab81283, ABcam, USA), anti-ERK (ab54230, ABcam, USA), anti-β-actin (ab11003, ABcam, USA), anti-RHOC (ab64659, ABcam, USA), anti-p-ERK (ab201015, ABcam, USA), ani-p-AKT (ab38449, ABcam, USA), at 4 °C overnight. After washing, the membrane was cultured with HRP-linked goat anti-rabbit Ab (Cell Signaling Technology). Blots were visualized by ECL (ECL Plus; Amersham Pharmacia Biotech, Uppsala, Sweden), on the basis of the manufacturer protocol. Results were captured digitally using a LAS1000 Lumino Image Analyzer (Fuji Photo Film, Tokyo, Japan).

Cells were lysed with RIPA buffer (Nacalai Tesque) supplemented with 0.1% bromophenol blue and 10% β-mercaptoethanol and denatured for 5 min. Cell lysates were then loaded onto a 6–10% polyacrylamide gel and subjected to SDS–PAGE. The separated proteins were transferred onto a polyvinylidene fluoride microporous membrane (Millipore, Burlington, MA, USA), and the membrane was blocked with 5% skim milk in tris-buffered saline with Tween 20. Respective primary and secondary antibodies (Supplementary Table S3) were used to detect specific proteins. The signals were detected using Immobilon Western Chemiluminescent Horseradish Peroxidase Substrate (Millipore). Signal intensities were measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA), and target protein levels were normalized to that of β-ACTIN and relative protein levels were calculated. Uncropped images of the western blotting membranes are shown in Supplementary Figures S10–S14.

MDCK II cells were seeded in poly-L-lysine-coated 24-well plates at a density of 2.1 × 10⁵ cells per well. The cells were cultured for 3 days, and the medium was changed every day to establish monolayer integrity. After specific treatments, monolayers were washed once with phosphate-buffered saline (PBS) and lysed with lysis buffer. After sonication, the cell extracts were boiled at 60 °C for 10 min, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene fluoride microporous membrane (Millipore, Billerica, MA), blocked with 5% skimmed milk (Megmilk Snowbrand, Sapporo, Japan) or setsuyakukun supporter (#DRC-BS500CH, DRC, Tokyo), probed with the appropriate primary antibody and HRP-conjugated anti-IgG secondary antibody, and detected by enhanced chemiluminescence (#07880-70, Nakalai Tesque). Images were visualized using X-ray film (RU-X, Fuji film) for Figs 4A and 5E, and using Sayaka Imager (DRC, Tokyo) for Fig. 6E. The band intensities of phospho-cofilin, occludin and actin were measured with Image J and normalized by actin.