SPP1/Osteopontin expression and a validation dilemma. SPP1 expression comparison of RNA and protein. A. Northern blot analysis. Eight μg of whole cell total RNA extracts were probed with a 1384 bp fragment of SPP1 (see methods) or 800 bp, Pst1 fragment of RPLP0 (36B4). Phosphorimaging was used for detection and quantitation. B. Western immunoblot analysis. Two independent preparations of each clonal cell line were used. Three mL of serum free-media from 3 × 105 cell equivalents was immunoblotted, using a goat anti-SPP1/osteopontin polyclonal antibody (antibody and recombinant protein were a gift from Dr. CM Giachelli) and a secondary rabbit anti-goat antibody (Pierce).