Supporting data pertaining to Cdc5-dependent phosphorylation of Nup60 and other novel meiotic targets. (A) Montage of a cell with Nup60-GFP, a nuclear basket nucleoporin, and Spc42-mCherry, a spindle pole body component, entering metaphase I arrest caused by cdc5-mn (UB29251). (B) Montage of a cells with Nup60-GFP, a nuclear basket nucleoporin, and Htb1-mCherry, a histone, in prophase I arrest (ndt80Δ) with PCUP1-CDC5-3xFLAG-10xHis uninduced (UB29129). Normalized DI values are indicated when calculated (see Fig. 3 H legend for description of normalization). For A and B, the white arrowheads in the “Fire LUT” images denote nuclei exhibiting Nup60-GFP detachment or nuclei from a relevant control at an equivalent time point. Scale bars, 2 μm. (C) FACS profiles of DNA content of meiotic time courses used for SWATH-MS proteomics in Fig. 4, A–D (Cdc5: YML3993, Cdc5KD: YML3994). Three independent replicates are shown for each strain. (D) Number of phosphopeptide precursors identified in each sample. The color scale represents the consistency of identification as a fraction over all runs. (E) Representative immunoblot of Cdc5 expression in cells treated as described in Fig. 4 A. Cells with ndt80Δ and PGAL1-CDC5-eGFP (YML3993) were induced to enter meiosis by transfer to SPM* and, after 7 h in SPM*, treated with 2 µM β-estradiol to initiate Cdc5 expression. Puf6 was used as a loading control. (F) Immunoblot for Nup60-GFP before (4 h in SPM) or during (7 h in SPM) metaphase I arrest for the strains imaged in panels 3C-D and S4A (cdc5-mn: UB29251, cdc20-mn: UB29253, and cdc5-mn cdc20-mn: UB29249). Hxk2 was used as a loading control. (G) Immunoblots for Nup60-GFP and Cdc5KD-3xFLAG-10xHis (UB29069) or Cdc5-3xFLAG-10xHis (UB29129) before (5 h in SPM) or after (6 h in SPM) treatment (either addition of copper or not) during prophase I arrest (ndt80Δ). The protein samples were collected from the strains imaged in 3F-G and S4B. Hxk2 was used as a loading control. (H) Immunoblots of Swi6-9myc and Cdc5 in cdc20-mn (YML8836) or cdc20-mn cdc5-mn (YML8837) strains induced to enter meiosis and arrest in metaphase I. Crm1 was used as the loading control. (I) Immunoblots for Slk19-9myc and Cdc5 in cdc20-mn (YML7800) or cdc20-mn cdc5-mn (YML7801) cells induced to enter meiosis and arrest in metaphase I. Crm1 was used as the loading control. For F–I, the brackets to the left of the blots denote apparent phosphoshifts. Source data are available for this figure: SourceData FS4.
Figure S4.

Supporting data pertaining to Cdc5-dependent phosphorylation of Nup60 and other novel meiotic targets. (A) Montage of a cell with Nup60-GFP, a nuclear basket nucleoporin, and Spc42-mCherry, a spindle pole body component, entering metaphase I arrest caused by cdc5-mn (UB29251). (B) Montage of a cells with Nup60-GFP, a nuclear basket nucleoporin, and Htb1-mCherry, a histone, in prophase I arrest (ndt80Δ) with PCUP1-CDC5-3xFLAG-10xHis uninduced (UB29129). Normalized DI values are indicated when calculated (see Fig. 3 H legend for description of normalization). For A and B, the white arrowheads in the “Fire LUT” images denote nuclei exhibiting Nup60-GFP detachment or nuclei from a relevant control at an equivalent time point. Scale bars, 2 μm. (C) FACS profiles of DNA content of meiotic time courses used for SWATH-MS proteomics in Fig. 4, A–D (Cdc5: YML3993, Cdc5KD: YML3994). Three independent replicates are shown for each strain. (D) Number of phosphopeptide precursors identified in each sample. The color scale represents the consistency of identification as a fraction over all runs. (E) Representative immunoblot of Cdc5 expression in cells treated as described in Fig. 4 A. Cells with ndt80Δ and PGAL1-CDC5-eGFP (YML3993) were induced to enter meiosis by transfer to SPM* and, after 7 h in SPM*, treated with 2 µM β-estradiol to initiate Cdc5 expression. Puf6 was used as a loading control. (F) Immunoblot for Nup60-GFP before (4 h in SPM) or during (7 h in SPM) metaphase I arrest for the strains imaged in panels 3C-D and S4A (cdc5-mn: UB29251, cdc20-mn: UB29253, and cdc5-mn cdc20-mn: UB29249). Hxk2 was used as a loading control. (G) Immunoblots for Nup60-GFP and Cdc5KD-3xFLAG-10xHis (UB29069) or Cdc5-3xFLAG-10xHis (UB29129) before (5 h in SPM) or after (6 h in SPM) treatment (either addition of copper or not) during prophase I arrest (ndt80Δ). The protein samples were collected from the strains imaged in 3F-G and S4B. Hxk2 was used as a loading control. (H) Immunoblots of Swi6-9myc and Cdc5 in cdc20-mn (YML8836) or cdc20-mn cdc5-mn (YML8837) strains induced to enter meiosis and arrest in metaphase I. Crm1 was used as the loading control. (I) Immunoblots for Slk19-9myc and Cdc5 in cdc20-mn (YML7800) or cdc20-mn cdc5-mn (YML7801) cells induced to enter meiosis and arrest in metaphase I. Crm1 was used as the loading control. For F–I, the brackets to the left of the blots denote apparent phosphoshifts. Source data are available for this figure: SourceData FS4.

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