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. 2019 Sep 2;132(17):jcs228601. doi: 10.1242/jcs.228601

Fig. 5.

Fig. 5.

Sirtuin 7 interacts with fibrillarin and Nop56. (A) GST and GST–SIRT7 pulldown assays were performed using whole-cell extracts prepared from HeLa cells or HeLa cells expressing either GFP–15.5kD, GFP–Nop56 or GFP–Nop58. 1% of extract used for each pulldown assay (Input) and 5% of the proteins eluted after GST–SIRT7 and after GST pulldown assays were submitted to 10% SDS-PAGE. Immunoblotting was performed using serum with specificity against Nopp140 and anti-nucleolin, anti-B23 and anti-fibrillarin antibodies for the pulldown assays performed using HeLa cells, and with anti-GFP antibody for the pulldown assays performed using HeLa cells expressing a GFP–protein fusion. (B) GST, GST–FIB and GST–Nop56 pulldown assays were performed using recombinant human sirtuin 7. (a) Ponceau Red staining of samples resolved by SDS-PAGE and transferred to nitrocellulose membrane. (b) Immunoblotting using anti-SIRT7 antibody. Lane 1: input control corresponding to 0.1 µg of recombinant sirtuin 7 and 10 µg of BSA. Lanes 2–5: buffer (lane 2, Beads) or the lysates containing GST (lane 3, GST), the GST–fibrillarin fusion (lane 4, GST–FIB) or the GST–Nop56 fusion (lane 5, GST–Nop56) were incubated with glutathione–agarose beads overnight and washed. The beads were then incubated for 2 h with 0.1 µg of recombinant sirtuin 7 and 10 µg of BSA, and washed. Lanes 6–9: buffer (lane 6, Beads) or lysates containing GST (lane 7, GST), the GST–fibrillarin fusion (lane 8, GST–FIB) or the GST–Nop56 fusion (lane 9, GST–Nop56) were incubated with glutathione–agarose beads for 1 h and washed. The beads were then incubated overnight with 0.1 µg of recombinant sirtuin 7 and 10 µg of BSA, and washed. (C) GST and GST–SIRT7 pulldown assays were performed using whole-cell extracts prepared from HeLa cells expressing either FIB–GFP, FIB/1-100–GFP, FIB/1-273–GFP or FIB/81-321–GFP. 1% of extract used for each pulldown assay (Input) and 5% of the proteins eluted after GST–SIRT7 and GST pulldown assays were resolved by 10% SDS-PAGE. Immunoblotting was performed using anti-GFP antibody. A schematic representation of domain structures of fibrillarin and fibrillarin truncation mutants is given on the right of the figure. The GAR, RBD and α-helix domains, and residue numbers are indicated above the diagram. Cropped immunoblots are shown in A and C. The asterisk corresponds to BSA and arrowheads point to the full-length GST fusion proteins in Ba.