Secondary antibodies conjugated to horseradish peroxidase

Neutrophils (10⁷ cells per condition) were lysed in 200 µl of RIPA buffer containing 150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1% Triton X-100, 1 mM EDTA, 1 mM PMSF, a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO), 1 mM sodium orthovanadate and 100 mM sodium fluoride. Proteins were separated on 8% bis-acrylamide gels, transferred to a nitrocellulose membrane, and blocked with 5% milk in TBST (50 mM Tris [pH 7.5], 150 mM NaCl, and 0.05% Tween) for 1 h at room temperature. Immunodetection was performed using antibodies against phospho-erk1/2 or erk1/2 (Santa Cruz Biotechnology, Dallas, TX), phospho-p38 (Thr180/Tyr182) (Cell Signaling Technology) and β-actin (Sigma-Aldrich, St. Louis, MO), followed by secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Dallas, TX). The blots were revealed by enhanced chemiluminiscence (ECL) in an Image Quant 300 cabinet (GE Healthcare Biosciences, PA, USA) following the manufacturer instructions. Images were analyzed with ImageJ software.

Lung tissue (200 mg) was homogenized in RIPA lysis buffer (100 ml PBS 1x pH 7.2, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, supplemented with protease inhibitors). Total protein was quantified using Bradford assay (Bio-Rad, Hercules, CA). Samples were subjected to SDS-PAGE electrophoresis, using a 15% acrylamide gel for CC10 and 10% acrylamide for Notch1. Seventy five μg of protein were loaded for CC10 and 50 μg for Notch1. They were transferred to PVDF membranes and blocked with 5% nonfat milk. The following primary antibodies were used: mouse anti-Muc5ac (1:2000, sc-21701, Santa Cruz Biotechnology), goat anti-Muc5b (1:2000, sc-135508, Santa Cruz Biotechnology), rabbit anti-CC10 (1:4000, ab40873, Abcam, UK), goat anti-Notch1 (1:2000, sc-6015, Santa Cruz Biotechnology), and Anti-Actin I-19 (1:2000, sc-1616, Santa Cruz Biotechnology, USA). Secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology) were used. Brain and stomach were used as positive controls. Water without antibodies was used as negative control. Proteins were detected by Pierce, ECL Western Blotting Substrate (Pierce Biotechnology, Rockford, IL). Images were film-captured. Density of band was measured using Fiji software (ImageJ 2.0.0-rc-54/1.51h) and protein amounts were normalized with the actin signal.

300,000 HT1080 stable cells expressing the ssGFP-FM4-FCS-hGH cargo were grown on 6-well plates. Secretion assays were performed in HBSS (Life Technologies) supplemented with 1% fetal bovine serum (Life Technologies) and 100 μg/ml cycloheximide (Sigma-Aldrich, St. Louis, MO). Incubations at 20 and 37 °C were performed in temperature-controlled incubators. Traffic was initiated by adding 1 μM D/D solubilizer (Clontech, Mountain View, CA), then at the indicated time points, chase media were collected and precipitated with 10% v/v trichloroacetic acid (Sigma-Aldrich, St. Louis, MO), washed in acetone, then dried. Cells were scraped and pelleted. Chase medium and cell extracts were separated by SDS-PAGE, then transferred onto nitrocellulose membrane for immunoblotting. After blocking with 5% w/v fat-free milk in PBS-T, the membranes were incubated with the appropriate primary antibodies. We used anti-Tag(CGY)FP (Evrogen, Moscow, Russia), anti-actin (Cell Signaling Technology, Danvers, MA), anti-GRASP55 (Proteintech, Rosemont, IL), anti-GAPDH (Sigma-Aldrich, St. Louis, MO). Then we used appropriate secondary antibodies conjugated to horse-radish peroxidase (Santa Cruz Biotechnology, Santa Cruz, TX) for chemiluminescence detection.