Figure 6. Naa50/San is not required for overall integrity of the cohesin complex.

(A) All subunits of the cohesin complex, but not Dalmatian/Sororin, were efficiently immunoprecipitated with endogenous Scc1 or with Myc-tagged Scc1 after depletion of Naa50/San. Co-immunoprecipitations with an anti-Scc1 antibody or with anti-c-Myc Magnetic beads (Invitrogen, Grand Island, NY, USA) were performed, respectively, using total protein extracts from Drosophila S2 cells or from S2 cells expressing a Myc-tagged Scc1. Both sets of cells were either treated with control RNAi or san RNAi before immunoprecipitation. (−), (+), (++), and (+++) corresponds, respectively, to 0, 1–9, 10–19, and >20 non-repeated peptides detected by LC-MS. R1 and R2 correspond to replica 1 and replica 2, respectively. None of the proteins shown in this analysis were detected in the negative controls (respectively, pre-immune serum or Drosophila S2 cells expressing an empty plasmid). For detailed LC-MS analysis see Supplementary Table 4. (B,C) Endogenous Scc1 was efficiently co-immunoprecipitated by Smc3-GFP after depletion of Naa50/San, both in actively dividing (B) or metaphase-arrested (C) cells. On the other hand, Dalmatian/Sororin (Dmt) was not co-immnunoprecipitated by Smc3-GFP in both conditions. Co-immunoprecipitation from total protein extracts from Drosophila S2 cells expressing GFP-tagged Smc3 (Smc3-GFP) and using anti-GFP coated Dynabeads. Protein extracts from Drosophila S2 cells transfected with an empty plasmid were used as a negative control. For metaphase arrest, Drosophila S2 cells were treated with 25 μM of colchicine for 12 hours. The mitotic index (% of phospho-H3 (pSer10) positive cells) of S2 cells treated or not with colchicine is shown in Supplementary Fig. 6.