Memcode reversible protein stain kit

In addition to eluates of the co-IPs, lysates of testes and epididymal sperm from five wild-type and five ChAc^Del/Del mice were individually solubilized with RIPA buffer (Thermo Fisher Scientific, Rockford, IL, USA) and analyzed by Western blotting. The proteins were denatured with NuPAGE LDS Sample Buffer (Life Technologies Corporation), separated on appropriate gels and transferred to polyvinylidene difluoride membranes (Merck KGaA, Darmstadt, Germany). The amount of protein sample was visualized using the MemCode Reversible Protein Stain Kit (Thermo Fisher Scientific). Membranes were blocked for 1 h at room temperature with 3% non-fat dried milk in PBS containing 0.1% Tween-20, and then, they were incubated with primary antibodies (chorein, PGK2, LDHC, ACAT1, IDH3A, ANXA1, ODF1, TOM20, and alpha-tubulin) and the appropriate secondary antibodies. Proteins were visualized using ECL Prime Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK), and images were recorded with a digital analyzer (FUSION-SOLO.7S.WL; Vilber-Lourmat, Marne-la-Vallée, France). ImageJ software version 1.52a (U.S. National Institutes of Health, Bethesda, ML, USA) was used for the densitometric analysis of protein bands. The results were standardized to alpha-tubulin. The Mann–Whitney U test was performed for statistical analysis using EZR in R Commander version 1.27 [14 (link)].

Ventricular tissue was collected at 7 weeks of age and homogenized in lysis buffer (150 mM sodium chloride, 1.0% Triton X‐100, 0.5% sodium deoxycholate, 0.1% Sodium dodecyl sulfate, 50 mM Tris, pH 8.0) using silica beads and a homogenizer (BioSpec). Lysates were measured using the Pierce BCA assay kit (Thermo Scientific #23225) and normalized to 10 µg total protein. Lysates were separated using polyacrylamide gel electrophoresis and gel was transferred to nitrocellulose (NC) membranes. Membranes were blocked in 5% BSA prepared in 1x TBS‐T. After blocking, NC membranes were incubated with primary antibodies Cx40 (1:1000, Cx40‐A, Alpha Diagnostic Intl Inc.), Connexin 43 (1:1000, Cx43, Sigma C6219), or Contactin 2 (1:1000, R&D Systems, AF4439). Membranes were incubated with secondary antibody goat anti‐rabbit IgG‐HRP (1:1000, Santa Cruz Biotechnology sc‐2004) or donkey anti‐goat IgG‐HRP (1:1000, Santa Cruz Biotechnology sc‐2020). Signals were detected using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific #34577). Total protein was determined using Memcode Reversible Protein Stain Kit (Thermo Scientific #24580).

ECs were grown to subconfluence and culture medium was replaced with fresh EGM-2 MV. After 18–24 h, CM was collected and passed through a 0.22-μm filter (Merck Millipore) and subsequently concentrated approximately 120-fold using Amicon Ultra-15 30 K centrifugal filter units and Amicon Ultra 0.5-mL 30 K centrifugal filters (Merck Millipore). LM-tumour cells were preincubated with anti-TLR2 and/or TLR4 antibodies (Biolegend) for 90 min, or with U0126 or BAY11-7082 for 60 min. Cells were lysed after stimulation of biglycan for 30 min (ERK) or 60 min (NF-κB). Equal amounts of total protein were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PDVF) membranes. Equal loading and transfer were confirmed by MemCode Reversible Protein Stain Kit (Thermo Scientific) for detection of biglycan in the CM of ECs. Western blotting was performed using antibodies listed in Supplementary Table S1 and an HRP-conjugated secondary antibody, as previously described10 (link)12 (link).

Protein expression was evaluated with western blotting according to methods previously described in detail [16] (link). In brief, tissues were homogenized in lysis buffer with protease and phosphatase inhibitors, and the proteins were extracted. The samples were boiled for 5 min in Laemmli sample buffer, separated using SDS-PAGE, and transferred to a polyvinylidene fluoride membrane. A chemiluminescence reagent was used to detect immunoreactive bands. ImageJ 1.38x software was used for semi-quantitative densitometric analysis of the bands. The density of each band was normalized to that of the tubulin band. Non-denatured and non-reduced samples were used for the detection for collagen IA and IIIA proteins. The intensities of collagen IA and IIIA bands were normalized to the intensity of a membrane protein band (MemCode Reversible Protein Stain kit; Thermo Fisher Scientific Inc., Waltham, MA). The antibodies were used at the following dilutions: anti-collagen IA (1∶100), anti-collagen IIIA (1∶250), anti-collagen IV (1∶500), anti-MCP-1 (1∶100), anti-IL-1β (1∶250), anti-αSMA (1∶1500), anti-fibronectin (1∶500), anti-p22^phox (1∶250), anti-NOX4 (1∶500), TGF-β1 (1∶250), anti-phospho Smad3 (1∶250), anti-total Smad3 (1∶1000), anti-TfR (1∶1000), anti-DMT1 (1∶250), anti-FPN (1∶500), anti-FTH (1∶250), anti-FTL (1∶250), and anti-tubulin (1∶1000).