Figure S4. The sir4 K1037R mutation... | Rockefeller University Press

Figure S4.

The sir4K1037R mutation affects telomere tethering but not Sir4 NE localization.(A and D) SUMO conjugates (A), Sir4-V53, or sir4K1037R-V53 (D) bound to chromatin adjacent to Tel6R was assessed by ChIP and qPCR analysis using antibodies directed against SUMO or the V5 epitope in asynchronous cultures of the indicated strains. Graphs represent at least three biological replicates. Error bars represent SEM. Asterisks: significant change relative to WT using a two-tailed Student’s t test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (B) Tethering of Tel6R in WT and sir4K1037R-V53 cells was examined as described in Fig. 6 A. (C) NE localization of Sir4-GFP and sir4K1037R-GFP was assessed as described in Extended Data Fig. S3 D. Bar graphs show the percentage of total GFP foci at the NE (left panel) and the average number of foci per nucleus (right panel) at the indicated cell cycle stage. Cells in anaphase/telophase show double the number of Sir4-GFP foci as seen in G1- and S-phase cells. Note, WT data shown are the same as those in Extended Data Fig. S3 D. Graphs in B and C represent data from at least three biological replicates. n = 50 cells/replicate/cell cycle stage. Error bars represent SD. Asterisks: significant difference relative to WT using a two-tailed Student’s t test. **, P ≤ 0.01. Ana/Telo, Anaphase/Telophase; Phos, phosphorylation.

The sir4^K1037R mutation affects telomere tethering but not Sir4 NE localization. (A and D) SUMO conjugates (A), Sir4-V5₃, or sir4^K1037R-V5₃ (D) bound to chromatin adjacent to Tel6R was assessed by ChIP and qPCR analysis using antibodies directed against SUMO or the V5 epitope in asynchronous cultures of the indicated strains. Graphs represent at least three biological replicates. Error bars represent SEM. Asterisks: significant change relative to WT using a two-tailed Student’s t test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (B) Tethering of Tel6R in WT and sir4^K1037R-V5₃ cells was examined as described in Fig. 6 A. (C) NE localization of Sir4-GFP and sir4^K1037R-GFP was assessed as described in Extended Data Fig. S3 D. Bar graphs show the percentage of total GFP foci at the NE (left panel) and the average number of foci per nucleus (right panel) at the indicated cell cycle stage. Cells in anaphase/telophase show double the number of Sir4-GFP foci as seen in G1- and S-phase cells. Note, WT data shown are the same as those in Extended Data Fig. S3 D. Graphs in B and C represent data from at least three biological replicates. n = 50 cells/replicate/cell cycle stage. Error bars represent SD. Asterisks: significant difference relative to WT using a two-tailed Student’s t test. **, P ≤ 0.01. Ana/Telo, Anaphase/Telophase; Phos, phosphorylation.