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. 2018 Apr 17;131(8):jcs211482. doi: 10.1242/jcs.211482

Fig. 8.

Fig. 8.

NCDN does not co-purify with snRNPs, while NCDN and SMN interact with Rab5 and colocalise with a subset of Rab5 vesicles within neurites of SH-SY5Y cells. (A) Incubation of whole-cell lysate from an SH-SY5Y cell line constitutively expressing NCDN–GFP with agarose beads conjugated to antibodies against the tri-methyl guanosine cap (Me3Gppp) of snRNAs (TMG beads) affinity purifies snRNPs as evidenced by the enrichment of the core snRNP protein SNRPN (detected with anti-SNRPN antibody, bottom row). The enriched snRNP fraction also contains SMN, which is essential for snRNP assembly. NCDN–GFP, however, does not co-enrich with snRNPs. Also shown is the core structure of mature snRNPs consisting of the heptameric Sm protein ring bound at the Sm-binding site of snRNA, as well as the characteristic tri-methyl guanosine Cap of snRNAs (Me3Gppp) at the 5′ end. (B) Affinity isolation of mRFP–Rab5 using RFP-Trap from cells co-transfected with plasmids to express mRFP–Rab5 together with NCDN–GFP, GFP–SMN or YFP alone co-enriches both NCDN–GFP (top row, detected with anti-GFP antibody, band is present in RFP-Trap lane but not Sepharose beads lane) and SMN-GFP (second row, detected with anti-GFP antibody, band is present in RFP-Trap lane but not Sepharose beads lane), but not YFP (third row, no band detected in RFP-Trap lane). Endogenous SMN (fourth row, detected with mouse anti-SMN) co-enriches with mRFP–Rab5 in all three samples. Detection of mRFP–Rab5 (bottom row, detected with anti-RFP antibody) confirms substantial enrichment of mRFP–Rab5 in all three samples. (C) Both GFP–SMN and NCDN–GFP partially colocalise with mRFP–Rab5 in a subset of mRFP–Rab5-containing vesicles in co-transfected SH-SY5Y cells (white signal in overlaid images, top row; yellow signal in colocalisation images, bottom row). (D) Enlargement of the boxed areas in C confirms that the colocalisation between SMN or NCDN and Rab5 occurs in punctate structures. Arrowheads identify areas of colocalisation. Colocalisation images were generated by Volocity using automatic thresholds on non-deconvolved z-sections before excluding values below 0.05. Images (excluding the colocalisation images) are single deconvolved z-sections. Scale bars: 7 µm.