Anti prx1

Cells were lysed with RIPA buffer containing a proteinase inhibitor cocktail (Fisher Scientific, Pittsburg, PA), sheared 10 times by passage through a 28-gauge needle, and centrifuged at 16,000 g for 30 min; the supernatants were normalized for protein concentration as determined by the Bradford method. Lysates were boiled for 5 min and resolved on 4–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels (Invitrogen, Carlsbad, CA). The blots were probed with the following primary antibodies from Cell Signaling Technology (Danvers, MA): p-STAT3-Tyr705, p-STAT3-Ser727, HKII, P70, and P70S6K. The following antibodies were from Santa Cruz Biotechnology (Dallas, TX): anti-actin, anti-Prx1 and anti-Prx3.

For immunofluorescence, decalcified samples were embedded in optimal cutting temperature compound (OCT) after dehydration, and cut at 8 μm sections. Slices were treated with 3% hydrogen peroxide for 15 min and blocked with heat inactivated goat serum before incubation in primary antibodies overnight at 4 °C. The following primary antibodies were used for detection: anti-PRX1 (1:100, Santa Cruz Biotechnology, Dallas, TX), anti-Osterix (1:200; Abcam, Cambridge, UK), anti-RUNX2 (1:200; Abcam, Cambridge, UK), and anti- Vimentin (1:100, Cell Signaling Technology, Boston, USA). After incubation with Alexa Fluor 488 IgG or 594 IgG (1:100 0, Invitrogen, USA) at room temperature for 1 hours, the cell nuclei were counterstained with DAPI (1 μg/mL, Sigma-Aldrich, USA). Immunofluorescence images were visualized and captured with a confocal laser scanning microscope (Nikon, TI2-E + A1 R, Japan).