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. 2003 Jul 28;100(17):9762–9767. doi: 10.1073/pnas.1732541100

Fig. 3.

Fig. 3.

Endogenous Nfu proteins are present in the cytosol, mitochondria, and nucleus of RD4 cells. (A) Western blot analysis of subcellular fractions of RD4 cell lysates by using α-Nfu 4333 and α-Nfu(MAP) 4337. Analysis of a nuclear protein (Oct-1) and a mitochondrial protein (Mn-SOD) are included for comparison. (B) Western blot analysis of RD4 triton lysate (lane 1) using α-Nfu 4333. In vitro translation products of pcDNA-nfu isoform I, pcDNA-nfu isoform II, and a control construct, pPoly(A)-luciferase, were shown for comparison. (C) RD4 cells were pulse-labeled with [35S]methionine and chased at the specified times. Immunoprecipitation of the triton lysates withα-Nfu 4333 indicated a two-step processing of Nfu isoform I. (D) Immunoprecipitation of endogenous Nfu in RD4 lysates, compared with immunoprecipitation of the in vitro translation products of nfu isoform I and II. As expected, in vitro translation of isoform I gives rise to a major protein of ≈28 kDa, whereas isoform II gives rise to a protein of ≈26 kDa. The shorter product of isoform II (≈22 kDa) may be the products of in vitro translation initiated at ATG3 (Fig. 1A).