Fe65 electrophoretic mobility shift due to phosphorylation is not detected in the nuclear fraction.

<p>A. Western blot analysis of Fe65 in cytoplasmic (C1) and nuclear (N1) fractions after incubation in the absence or presence of alkaline phosphatase. Two different exposure times are shown for optimal visualization of Fe65 in the different fractions. B. Western blot analysis of Fe65 in cytoplasmic (C1), nuclear (N1) and cytoplasmic and nuclear mixed (C1 + N1) fractions after incubation in the absence or presence of alkaline phosphatase. C. Western blot analysis showing the effect of alkaline phosphatase treatment of cytoplasmic fractions on endogenous Fe65 and p97Fe65-TAP (C2), p60Fe65-TAP (C3), or the deletion mutants ΔWWFe65-TAP (C4) and ΔPTB2Fe65-TAP. D. Representative western blot analysis of lysates from SK-N-AS cells overexpressing the different Fe65 constructs lacking the TAP tag. The results are from the same blot. Different exposure times are shown for optimal visualization.</p

Subcellular distribution of overexpressed TAP-tagged and endogenously expressed Fe65.

<p>A. Representative western blot analysis of endogenously expressed Fe65 in cytoplasmic (C, lane 1) and nuclear (N, lane 2) fractions obtained from SH-SY5Y cells. B. Representative western blot analysis comparing levels of TAP-tagged Fe65 (p97Fe65 TAP) with endogenously expressed Fe65 in cytoplasmic (C) and nuclear (N) fractions obtained from SH-SY5Y cells. Lamin B and βIII-tubulin were used as markers for nucleus and cytoplasm, respectively. C. Representative western blot analysis of APP levels obtained after Fe65 TAP pull-down and TEV cleavage in SK-N-AS cells transfected with cDNA coding for Fe65 TAP and APPwt. D. Schematic illustration of Fe65 full-length (p97Fe65), the shorter isoform p60Fe65, and two deletion mutants fused to the TAP tag. E. Representative western blot analysis of cytoplasmic (C) and nuclear (N) fractions from SH-SY5Y cells overexpressing the different TAP-tagged Fe65 constructs. Two different exposure times are shown for optimal visualization of TAP-tagged Fe65 constructs in cytoplasmic and nuclear fractions, respectively. Note, the N-terminal Fe65 antibody (E-20) binds to the prot A sequence in the TAP tag leading to the visualization of the N-terminally truncated Fe65 constructs. F. Representative western blot analysis of cytoplasmic (C) and nuclear (N) fractions from SK-N-AS cells overexpressing the different TAP-tagged Fe65 constructs. Note, only secondary anti-goat IgG was used for visualization of TAP- tagged Fe65. G. Schematic illustration of Fe65 full-length (p97Fe65), the shorter isoform p60Fe65, and two deletion mutants lacking the TAP tag. H. Representative western blot analysis of cytoplasmic (C) and nuclear (N) fractions from SH-SY5Y cells overexpressing p97Fe65 and ΔPTB2Fe65. Two different exposure times are shown for optimal visualization of p97Fe65 and ΔPTB2Fe65 in cytoplasmic and nuclear fractions, respectively. I. Representative western blot analysis of cytoplasmic (C) and nuclear (N) fractions from SK-N-AS cells overexpressing ΔWWFe65 (C1 and N1) and ΔPTB2Fe65 (C2 and N2). For optimal visualization of Fe65 in cytoplasmic and nuclear fractions, the exposure time for nuclear fractions is 8 times longer than for the cytoplasmic fractions. Shown are representative images of three independent experiments.</p

Inhibition of ADAM10 or γ-secretase decreases the abundance of endogenous Fe65 in the nuclear fraction.

<p>A. Representative western blot analysis showing the levels of endogenously expressed Fe65 in the cytoplasmic and nuclear fraction of SH-SY5Y cells after treatment with the ADAM10 inhibitor GI254023X (A10inh), the γ-secretase inhibitor DAPT and the non-selective ADAM inhibitor Batimastat. The levels of sAPPα in the medium and APP in the cell lysate are shown for comparison. Lamin B and βIII-tubulin were used as markers for nucleus and cytoplasm, respectively. B. Relative abundance of Fe65 in the nuclear fraction (presented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173888#pone.0173888.g002" target="_blank">Fig 2C</a>). *P<0.05, **P<0.01, compared to control, n = 4.</p

ΔPTB2Fe65 differ in its subcellular localization from p97Fe65 and ΔWWFe65.

<p>Representative immunofluorescence microscopy images of SK-N-AS cells expressing p97Fe65-TAP (top row), ΔWWFe65-TAP (middle row), or ΔPTB2Fe65-TAP (bottom row). Fe65-TAP constructs were labeled with Alexa Fluor 488-conjugated anti-goat antibody. Cell nuclei were stained with DRAQ5. p97Fe65-TAP and ΔWWFe65-TAP were mainly localized in the nucleus whereas ΔPTB2Fe65-TAP was preferentially distributed to the cytoplasm. Note, TAP-tagged Fe65 was detected by the secondary anti-goat antibody alone due to the protein A domain. Scale bar 10 μM. Shown are representative images of three independent experiments.</p