Biochemical and functional demonstration of sIL6R in purified NPA from MEFs CM. (A) Immunodetection of sIL6R in NPA-positive fractions. Purified murine sIL6R or NPA at different stages of purification (Figure S2) were loaded on a 4% to 12% gradient gel, immunoblotted onto PVDF membrane, and probed with an antibody specific for murine IL6R. Reactive bands were detected with an HRP-coupled antigoat Ig antibody. Lane 1, molecular weight markers; lane 2, recombinant sIL6R, 20ng; lane 3, Superose-12 active pool A; lane 4, Mono-Q active pool A. This blot is representative of 2 separate experiments. (B) Selective immunodepletion of NPA by anti-IL6R antibody. Crude NPA-containing conditioned medium (CM) or semipurified NPA preparations (MonoQ A and B, Figures S1,S2) were preincubated with vehicle (), anti-IL6R antibody (▨), or anti-EGFR antibody (). The immune complexes were depleted from the samples using protein G-Sepharose as described in “Neutralization of NPA,” and the supernatants were assayed for NPA. Data are expressed as total NPA and have been adjusted for the small dilution factor resulting from antibody immunodepletion. All samples were tested in triplicate. Error bars represent SE.