Supersignal west dura extended duration substrate

E. faecalis cultures were grown for 16 h at 37°C. The spent supernatant was then treated with 5% TCA for 48 h at 4°C. Precipitated proteins were centrifuged at 10,000 rpm for 10 min, and the supernatant was then discarded. This step was repeated twice in order to ensure that all TCA was removed prior to solubilization in 6 M urea. TCA-precipitated proteins were separated by SDS-PAGE, and Western blotting was performed using a rabbit polyclonal anti-EntV antibody at a concentration of 1:1,000 and a horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody at a concentration of 1:10,000. The Western blots were processed using Thermo Fisher Scientific SuperSignal West Dura extended-duration substrate and imaged using the GE ImageQuant LAS4000 mini imaging system. To generate anti-EntV serum, full-length protein was recombinantly expressed from strain CEGE1 (15 (link)). Cell lysates were run on an SDS-PAGE gel, and the overexpressed protein was excised and sent to Cocalico Biologicals, Inc., for antibody generation.

Proteins were isolated and analyzed by immunoblotting. Briefly, the protein concentration in the samples was measured, samples were prepared in SDS and sample loading buffer, and heated for 10 min at 95 °C. Proteins were separated using 10% SDS-PAGE and immunoblotted onto membranes. The membranes were blocked with 1% bovine serum albumin (BSA) for 1 h and incubated with primary antibodies, including those against Nogo-A (Abcam, catalog no. ab62024), CHOP (Santa Cruz, catalog no. sc-71136), β-actin (Cell Signaling Technology, catalog no. 8457s), and GAPDH (Cell Signaling Technology, catalog no. 2118), overnight at 4°C. After a 1 h incubation with HRP-labeled secondary antibodies (Anti-rabbit-HRP, Cell Signaling Technology, catalog no. 7074s and Anti-mouse-HRP, Cell signaling Technology, catalog no. 7076s), the proteins were detected using enhanced chemiluminescence (ECL, Super Signal West Dura Extended Duration Substrate, catalog no. 34076) in an Amersham Imager 680 (GE Healthcare, Life Sciences). Blots were quantified using ImageJ software.

mProx were lysed in RIPA buffer containing phosphatase inhibitors (SIGMA-Aldrich). The nuclei extraction of mProx cells was performed with a Nuclear Extract Kit (Active Motif, Japan) according to the manufacturer’s protocol. Protein levels in cell and nuclear lysates were quantified using the Lowry method with a Bio-Rad DC™ protein assay kit (Bio-Rad Laboratories, U. S.), after which samples were prepared in cracking buffer such that each sample had the same amount of protein. Protein separation was performed on a polyacrylamide gel (gradient gel containing 10–20% acrylamide), and the blots were transferred to nitrocellulose membranes (Immun-Blot PVDF Membrane; Bio-Rad Laboratories), after which the membranes were hybridized with a peroxidase-labeled secondary antibody. SuperSignal™ West Dura Extended Duration Substrate was used to create a fluorescence signal, which was then viewed with an Image Quant LAS 4000 (GE Healthcare Life Sciences, US). Signal intensities were corrected with endogenous control proteins using Image J software (NIH, US).