Westar supernova detection kit

FDB muscles were homogenized using an electric homogenizer (Fluko, Shanghai, China) in a lysis buffer containing in mM: 20Tris-HCl (pH 7.5), 1% Triton X-100, 2 EDTA, 20 NaF, 1 Na₂P₂O₇, 10% glycerol, 150 NaCl, 10 Na₃VO₄, 1 PMSF and protease inhibitors (Complete™, Roche Applied Science). Proteins were separated using SDS-PAGE and transferred to PVDF membranes. The following primary antibodies and their dilutions were used as follows: Total NF-κB total p65 subunit (1:1000; Cell Signaling); p-Ser536-p65 (1:1000; Cell Signaling); p47^phox (1:5000; Sigma); p-p47^phox (pSer359) (1:5000; Sigma); gp91^phox (1:2000; BD Transduction); p-p38 (thr180/tyr182) (1:2000; Santa Cruz); p38 (1:2000; Santa Cruz); Phospho-ERK1/2(Thr202/Tyr204)(1:2000; Cell Signaling); ERK 1/2 (1:2000; Santa Cruz); α-Tubulin (1:5000; Cell Signaling); anti-mouse IgG-HRP (1:20,000; Santa Cruz); anti-rabbit IgG-HRP (1:30,000; Thermo Scientific Pierce). The protein bands in the blots were visualized using a WESTAR Supernova detection kit (Cyanagen, Bologna, Italy) and ChemiDoc™ MP System (Bio-Rad, USA). The intensity of the bands was determined with ImageJ densitometry analysis.

The fdb and soleus samples were homogenized with an electric homogenizer (Fluko, Shanghai, China) in a lysis buffer containing 20 mM Tris–HCl (pH 7.5), 1% Triton X-100, 2 mM EDTA, 20 mM NaF, 1 mM Na₂P₂O₇, 10% glycerol, 150 mM NaCl, 10 mM Na₃VO₄, 1 mM PMSF, and protease inhibitors (Complete^TM, Roche Applied Science). Proteins were separated using SDS-PAGE and transferred to PVDF membranes. The following antibodies and their dilutions were used: MCU (1:2,000; HPA016480, Sigma), MICU1 (1:2,000; HPA037480, Sigma), and TOM20 (1:10,000; ab186735, ABCAM). The protein bands in the blots were visualized using a WESTAR Supernova detection kit (Cyanagen, Bologna, Italy), super-resolution, and, further, ChemiDoc^TM MP System (Bio-Rad, United States). The intensity of the bands was determined with ImageJ densitometry analysis.

To evaluate the binding of vWbp to PPG and LTA, 5 μg of each compound was dotted onto a PVDF membrane (BioRad). After overnight incubation at 4 °C with 5% skim milk (w/v) in PBST, the membrane was treated for 1 h at 22 °C with 2 µg/mL of vWbp. The membrane was incubated with a mouse polyclonal vWbp antibody (1:5000) in 2% (w/v) skim milk for 1 h at 22 °C. Following several washings with PBST, the membrane was treated with 45 min at 22 °C with an HRP-conjugated rabbit anti-mouse IgG (1:1000) in 2% (w/v) skim milk and dot blots developed using the Westar Supernova detection kit (Cyanagen srl, Bologna, Italy). An ImageQuant™ LAS 4000 mini-biomolecular imager (GE Healthcare) was used to capture images of the spots. The signal intensities were quantified with ImageJ and plotted on GraphPad Prism.