Chemiluminescence detection reagent

TSP or purified proteins were separated using 10–12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under the reducing condition, and the proteins on the gels were either stained with 0.25% Coomassie blue R-250 (AMRESCO, cat. no: 6104-59-2, Zottegem, Belgium) solution or analyzed by Western blotting using mouse anti-His (Qiagen, Valenica, CA, USA), mouse anti-LIF (abcam, cat. no., ab34427, Cambridge, UK), mouse anti-GFP (Clontech, cat. No., 632381, CA, USA), rat anti-HA (Roche, cat. No., 1583 816, Basel, Switzerland), mouse anti-IL6 (abcam, cat. no., ab9324, Cambridge, UK), and anti-CBD (Bioapp, Pohang, Korea) antibodies. The horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG (Bethyl, cat. no., A90-146P, TX, USA), and goat anti-rat IgG (Bethyl, cat. no., A110-105P, TX, USA) were used as secondary antibody. Bands on the immunoblots were detected using chemiluminescence detection reagents (GE healthcare, Illinois, USA), and images were captured with a LAS 4000 image capture system (Fujifilm, Tokyo, Japan).

FACS-sorted green cells and single-cell islet suspension from diabetic and recipient mice isolated islets were lysed in RIPA buffer (1% triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 mM NaCl, 0.01 M Sodium Phosphate, pH 7.2). Fifteen micrometers protein after bradford quantification was loaded on 12% SDS-page to transfer onto a nitrocellulose membrane. Membranes were blocked with 1% BSA in PBS and probed with primary antibodies (see Table 1) at 4 °C overnight. The HRP-labeled secondary antibody was then probed for 30 min at RT. Membranes were finally stained with Chemiluminescence detection reagent and images were captured on the gel documentation system (GE Healthcare). Densitometric protein expression was measured from pooled cell extracts from 3 mice in duplicates, and fold changes with SD were calculated using Fiji software.

HUVECs grown in complete endothelial cell media were plated in tissue culture-treated 6-well plates at a density of 360,000 cells/well then serum-starved for 24 hours. The peptide SP2024 was added at 10 or 50 µM for 90 min. VEGF (Cell Signaling Technology, Inc., Danvers, MA) was then added at 20 ng/mL for 10 min. The reaction was stopped by adding cold PBS and lysis buffer (150 mM NaCl, 1 mM EDTA, 100 µL/ml protease inhibitors (Sigma, St. Louis, MO), 10 µL/ml phosphatase inhibitors (Sigma, St. Louis, MO) and 1% Triton) for 2 hours, and the cells were recovered by scraping. Cell lysates were spun at 14,000 g for 15 min to remove cell membranes and debris, separated by SDS-PAGE, and transferred to nitrocellulose blots (Invitrogen, Carlsbad, CA). Membranes were blocked for 1 hour with 5% milk and 1% BSA in TBST and probed with antibodies of interest in milk including anti-pVEGFR2, anti-phosphorylated PhospholipaseCγ (anti-pPLCγ), anti-PLCγ, and anti-GAPDH (Cell Signaling Technology, Inc). The next day, secondary antibodies were added at 1∶2000 dilutions, and protein bands were visualized with chemiluminescence detection reagent (GE Healthcare, United Kingdom). Blots were then stripped and probed for additional antibodies. Experiments were repeated at least once. Each blot shown is representative of one complete experiment.

The antibodies and chemicals were applied: anti-hepatitis B Virus X antigen (Abcam, Burlingame, CA, USA; 1:1000); anti-EZH2, clone AC22 (Cell Signaling; 1:1000); anti-HMGA2 (Abcam; 1:1000); and anti-β-actin (Sigma; 1:5000); Chemiluminescence detection reagent (GE, Piscataway, NJ). The experimental procedure refers to the previous report [11 (link)].