Figure 1.

α-COP co-immunoprecipitates Nucleolin. (a) Representative Western blot showing immunoprecipitations from HEK-293TT whole cell lysate with either rabbit polyclonal (CST NCL) or mouse monoclonal (SC NCL) antibodies. Immunoblotting with antibody against Nucleolin demonstrates immunoprecipitation with both antibodies compared to IgG control (mixed mouse and rabbit IgG). Blotting with antibodies against COPI complex subunits shows co-immunoprecipitation of α-COP and ε-COP but not β-COP. (b) Whole cell HEK 293TT lysates were split and treated with RNAse A followed by immunoprecipitation of Nucleolin. A representative Western blot shows that in both control and RNAse treated samples, α-COP co-immunoprecipitates with Nucleolin. Successful degradation of RNA was confirmed by conversion to cDNA followed by PCR. Multiple primer sets demonstrate that signal is only present in the untreated samples and absent from the samples treated with RNase. (c) Representative Western blot from fractionated 293-TT cells showing the localization of Nucleolin and α-COP in the α-tubulin positive cytoplasm (C) and the Histone H3 positive nuclear fraction (N). Representative Western blot showing the results of Nucleolin immunoprecipitation from cytoplasmic (C) and nuclear (N) fractions. As expected, the Nucleolin primarily co-precipitates α-COP from the cytoplasm. (d) Proximity ligation assay was performed on fixed mouse neuroblastoma cells (N2A). Negative control slides (mouse monoclonal α-COP antibody alone) show clean PLA background (red). Positive control coverslips combined mouse monoclonal α-COP antibody and rabbit polyclonal β′-COP, resulting in a strong PLA signal with predominantly Golgi localization (white arrowheads) as expected from COPI coatomer localization. Finally, experimental coverslips combined the mouse monoclonal α-COP with rabbit polyclonal Nucleolin antibody, resulting in a clear cytoplasmic PLA signal. Nuclei are visualized with DAPI.