Raw data for Western Blot in "Excess Neuropeptides in Lung Signal through Endothelial Cells to Impair Gas Exchange"
Description
Files here are raw data for all Western Blot images appeared in the research article titled "Excess Neuropeptides in Lung Signal through Endothelial Cells to Impair Gas Exchange". The file name corresponds to the panel number and the protein that was tested. All images was acquired by a LI-COR Biosciences imaging system and these files could be opened with Image Studio Lite software.
Files
Steps to reproduce
P22 mouse lung tissues were collected in RIPA buffer supplemented with Complete Protease Inhibitor Cocktail tablets (Roche) and PhosSTOP Phosphatase Inhibitor Cocktail tablets (Roche). Protein samples were then homogenized in a QIAGEN TissueLyser II. Protein concentrations were measured by PierceTM BCA Protein Assay kit (Thermo Scientific). An estimated ~10ug protein sample was loaded in each well and ran on a 4%–12% SDS-PAGE gel (Invitrogen), followed by transfer to PVDF membranes. PVDF membranes were blocked in TBST (0.1% Tween-20) with 5% BSA for 1.5 hours, incubated in primary antibodies overnight, and then incubated in the secondary antibody for 1 hour. All imaging and quantification were performed using the Image Studio Lite software system (LI-COR Biosciences). The following primary antibodies were used: CLDN5 (Invitrogen, 4C3C2, 1:2000), CDH5 (BD, 11D4.1, 1:1500) and β-actin (Novus Biologicals, NB600501, 1:5000). The secondary antibody used were: IRDye 680RD donkey anti–mouse IgG (LI-COR, 925-69072, 1:10,000) and IRDye 680LT goat anti-rat IgG (LI-COR, 925-68029, 1:10,000). Three biological replicates were performed for each antibody.