Appearance and localization of phosphorylated variants of the high molecular weight neurofilament protein in NB2a/d1 cytoskeletons during differentiation

Abstract

We used immunoblot and immunocytochemical methodologies to characterize the appearance and intracellular localization of the high molecular weight neurofilament subunit (NF-H) within the Triton-insoluble cytoskeleton during the first 5 days of differentiation of mouse NB2a/d1 neuroblastoma cells. Hypophosphorylated and partially phosphorylated forms of NF-H were detected in cells before and throughout differentiation. By contrast, some extensively phosphorylated forms of NF-H were first detected on the third day of differentiation and at least one additional 200 kDa isoform was visualized in cytoskeletons only after five days of differentiation. Extensively phosphorylated forms of NF-H were restricted to axonal neurites; by contrast, hypophosphorylated and partially phosphorylated forms of NF-H were present throughout undifferentiated and differentiated cells.