Anti smad1 antibody

BMSCs were treated with SFM (DMEM low glucose), 30 ng/mL BMP-2, 0.1 uM FTY720 or both for one hour. Then, samples were put on ice for 10 min, followed by centrifugation at 5000 rpm for 3 min. The supernatant was removed. Cells were lysed in a lysis buffer (Santa Cruz Biotechnology, Dallas, TX, USA) for 30 min, centrifuged at 4°C at 10,000 rpg for 10 min, and the supernatant was collected and run on a 4% acrylamide gel (Biorad, Hercules, CA, USA) for 30 min at 70 V and then 2 hours at 110 V. Proteins were transferred to a 45 um nitrocellulose membrane (Biorad). Finally, the membrane was blocked with 5% BSA (Santa Cruz Biotechnology) for 40 min on a shaker, then washed in 1× Tris buffered saline. The membrane was probed for either total smad-1 protein using anti-smad-1 antibody (Abcam, Cambridge, MA, USA) or phosphorylated smad-1 protein using phosphor smad-1 (ser206) mAB (Cell Signaling Technology, Danvers, MA, USA) and phosphor smad-1 (ser463/465) mAB (Cell Signaling Technology). Actin was used as a loading control. Protein was visualized using AlexaFLuor 680 labeled IgG (Invitrogen Life Technologies). Blots were imaged and quantified using a LiCor Odyssey (Licor Biosciences, Lincoln, NE, USA).

The tail tissues were fixed in 4% paraformaldehyde for 72 hours and then stored in 70% ethanol before µCT measurement or histological processing. A µCT35 desktop cone‐beam scanner (Scanco Medical) was used to scan the mouse tail at a resolution of 12 µm with a 55 kVp source and a 145 µAmp current. We performed three‐dimensional (3D) reconstruction on Co4‐Co7 vertebra. After CT reconstruction, we measured the intervertebral height of punctured disc normalized to adjacent vertebral body lengths using ImageJ. For comparison, the intervertebral heights of intact discs were also measured (n = 5).
After µCT examination, the tail tissues were decalcified with 10% formic acid for 3 weeks and then processed with a tissue processor (Excelsior™ AS Tissue Processor). The tail tissues were dehydrated with graded ethanol, immersed with xylene and embedded in paraffin. Serial sectioning was performed at thickness of 3 µm within the midsagittal region of the intervertebral disc. The sections were stained with Alcian blue & Orange G staining for histological analysis. Immunohistochemistry (IHC) was performed as previously reported,15 with sections incubated with 1:50 anti‐VEGF antibody (R&D Systems) or 1:200 anti‐Smad1 antibody (Abcam).