Whole femurs were analyzed as previously described.12 (link), 59 (link) Cells were cultured in 75 cm2 flasks for 12 days before treatment. 4 mL culture medium was filtered through a 50KDa ultra centrifugation column (EMD Millipore) and dried overnight in a Savant SpeedVac system (Thermo Fisher Scientific, MA, USA). Whole cell lysates were extracted using T-Per lysis buffer containing protease inhibitors cocktail, and nuclear and cytoplasmic extracts using NEPER™ Nuclear and Cytoplasmic Extraction kit (Thermo Fisher Scientific). Whole cell extracts and cytoplasmic lysates were filtered through a 50KDa ultra centrifugation size exclusion column and dried overnight.
Proteins were fractionated on 4-12 Bis-Tris minigels (Thermo Fisher Scientific), transferred on 0.22 PVDF membranes (Bio-Rad Laboratories) and probed with goat polyclonal anti-FGF23 (Immutopics), rabbit polyclonal MBS854462 anti-FGF23 (MyBioSource, CA, USA), rabbit polyclonal ab9485 anti-GAPDH, rabbit polyclonal ab2185 anti-HIF1α, rabbit polyclonal ab63766 anti-TATA binding protein TBP, and goat polyclonal ab8229 anti- β-actin antibodies (abcam). Antigen antibody complexes were visualized by immunoblotting using anti-goat and anti-rabbit IR fluorescent antibodies on an ODYSSEY® CLx system (LI-COR, NE, USA).
Proteins were fractionated on 4-12 Bis-Tris minigels (Thermo Fisher Scientific), transferred on 0.22 PVDF membranes (Bio-Rad Laboratories) and probed with goat polyclonal anti-FGF23 (Immutopics), rabbit polyclonal MBS854462 anti-FGF23 (MyBioSource, CA, USA), rabbit polyclonal ab9485 anti-GAPDH, rabbit polyclonal ab2185 anti-HIF1α, rabbit polyclonal ab63766 anti-TATA binding protein TBP, and goat polyclonal ab8229 anti- β-actin antibodies (abcam). Antigen antibody complexes were visualized by immunoblotting using anti-goat and anti-rabbit IR fluorescent antibodies on an ODYSSEY® CLx system (LI-COR, NE, USA).