Sc 59886

As described previously (Kang et al. 2013 (link)), cells were trypsinized and lysed by Pro-Prep protein extraction kit (iNtRON Biotechnology, Seongnam, Korea). Equal amounts of protein extracts (20 &g) were separated by sodium dodecyl sulfate–polyacrylamide gel electro­phoresis and transferred to a nitrocellulose membrane (Invitrogen). Blots were blocked with 5% nonfat dry milk at room temperature. The blots were incubated with antibodies specific for angiopoietin-1 (ab183701, Abcam), angiopoietin-2 (ab155106, Abcam), phosphorylated Tie2 (Tyr992, #4221, Cell Signaling Technology), Tie2 (sc-9026), thrombospondin-1 (sc-59886) and &-actin (Santa Cruz Biotechnology) at specific dilution, followed by incubation with peroxidase-labeled secondary antibodies. Immunoreactive proteins were visualized using an enhanced chemiluminescence detection kit (Santa Cruz Biotechnology). Blot images were captured using ImageQuant LAS 4000 biomolecular imager (GE Healthcare Life Sciences). Blot intensities were quantified using ImageJ 1.48v software (http://imagej.nih.gov/ij) (Schneider et al. 2012 (link)).

After cells were lysed in RIPA buffer (Beyotime, Shanghai, China) containing 1 mmol/L PMSF (Beyotime) and 0.1% protease inhibitor cocktail (Roche, Basel, Switzerland) for 30 minutes at 4℃, protein concentrations were determined (BCA Protein Assay Kit, Fisher Scientific Waltham. MA, USA). Protein samples (30 μg) were then separated by 10% SDS‐PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA,USA). Membranes were blocked in 5% non‐fat dried milk and then incubated at 4℃ overnight with primary TSP‐1 (1:1000, sc‐59886, Santa Cruz), GAPDH (1:2000, 10494‐1‐AP, Proteintech), MMP9 (1:1000, 10375‐2‐AP, Proteintech) and VEGF (1:1000, MAB293, R&D Systems) followed by incubating with horseradish peroxidase‐conjugated secondary antibody for 2 hours at room temperature. Protein bands were visualized using an enhanced chemiluminescence Amersham^TM ECL Plus Western blotting detection system (GE Healthcare, Amersham, UK).GAPDH was used as internal loading control.

The Western blot procedure was consistent with our previous work and detailed elsewhere (Hüttemann et al., 2012 (link), 2013 (link); Lee et al., 2012 (link); Malek et al., 2013 (link)). Samples were loaded onto 7.5% (TSP-1, PGC-1α, PGC-1β, and ADAMTS-1) or 12% TGX pre-cast gels (Bio-Rad, Hercules, CA, USA).
The mouse monoclonal primary antibodies used were TSP-1 (1:500, sc-59886, Santa Cruz Biotechnology, Inc), CD47 (1:500; 3847-1, Epitomics), PGC-1β (1:100; sc-373771, Santa Cruz Biotechnology, Inc), α-tubulin (1:2,000, ab11304, Abcam), ADAMTS1 (1:500; sc-47726, Santa Cruz Biotechnology, Inc), and GAPDH (1:2,000, ab9484, Abcam). The polyclonal primary antibodies used were VEGF (1:500, sc-507, Santa Cruz Biotechnology, Inc), VEGFR2 (1:500; 2479, Cell Signaling), FoxO1 (1:200; 2880, Santa Cruz Biotechnology, Inc), Anti-TFAM (1:1,000; ab131607, Abcam), and PGC-1α (1:1,000; AB3242, Millipore). The secondary antibodies used were goat anti-mouse IRDye (1:30,000) and goat anti-rabbit IRDye (1:30,000) purchased from Li-Cor Biosciences. Loading control for target proteins were normalized to α-tubulin or GAPDH. Quantification of bands were analyzed with the Odyssey software program (Li-Cor Biosciences).