EMBO/soda-nel huggingface dataset:
The cold response regulator CBF1 promotes Arabidopsis hypocotyl growth at ambient temperatures (2020)
Figure 6

Figure 6. CBF1 regulation of PIF4 and PIF5 protein abundance is modulated by low temperatures.

Phenotypes of Col, two cbf1 mutants (cbf1-1 and cbf1-2) and two CBF1-OE lines (CBF1-myc and CBF1-flag) grown under LD conditions at 22°C, 17°C and 4°C, respectively. The seeds of different genotypes were germinated under LD (16-h-light/8-h-dark photoperiod) conditions at 22°C for 2 d, respectively, and then grown under LD conditions at 22°C, 17°C, or 4°C, for additional 6 d. Scale bar = 1 mm.

Hypocotyl lengths of Col, two cbf1 mutants (cbf1-1 and cbf1-2) and two CBF1-OE lines (CBF1-myc and CBF1-flag) grown under LD conditions at 22°C, 17°C or 4°C. Error bars represent SD from 10 seedlings. ***P < 0.001 (two-tailed t-test) for the indicated genotype compared with Col. NS, not significant.

The ratios of hypocotyl lengths at 17°C versus 22°C for the indicated seedlings grown under LD conditions. Error bars represent SD from 10 seedlings. ***P < 0.001 (two-tailed t-test) for the indicated genotype compared with Col.

qRT-PCR assays showing the CBF1 transcript levels in Col seedlings grown under LD conditions at 22°C, 17°C and 4°C. Col seeds were germinated under LD conditions at 22°C for 2 d, and then grown under LD conditions at 22°C, 17°C or 4°C for additional 6 d. The samples were collected at ZT8, and then subjected to qRT-PCR assays. Error bars represent SD of three technical replicates. Different letters represent significant differences by one-way ANOVA with Duncan's post hoc test (P < 0.05).

E, F Immunoblots showing the protein levels of PIF4 (E) and PIF5 (F) in Col, cbf1-1, cbf1-2, and CBF1-myc seedlings grown under LD conditions at 22°C, 17°C and 4°C. The seeds of different genotypes were germinated under LD conditions at 22°C for 2 d, and then grown under LD conditions at 22°C, 17°C or 4°C for additional 6 d. The samples were collected at ZT20, and then subjected to immunoblotting. Anti-RPN6 was used as a sample loading control. Numbers below the immunoblots indicate the relative band intensities of PIF4 or PIF5 normalized to those of loading control, respectively. The ratio of the first band was set to 100 for each blot.
Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and hydrolysis (2014)
Figure 1

Generation of Ser65‐phosphoUb

Schematic representation of PINK1 variants phosphorylating Ub at Ser65. hPINK1, human PINK1; TcPINK1, Tribolium castaneum PINK1; PhPINK1, Pediculus humanus corporis PINK1.
Phos‐tag gel following complete conversion of Ub to phosphoUb, using GST‐PhPINK1. A Ser65 mutant Ub is not modified.
ESI‐MS analysis of pure Ub (left, mass 8,565.65 Da, reference 8,565.80 Da) and purified phosphoUb (right, mass 8,645.61 Da, reference 8,645.80 Da). The difference of 80 Da corresponds to one phosphate group.
AQUA analysis of PhPINK1‐phosphorylated Lys63‐linked hexaUb. The Lys63 peptide (K63) is modified by Ser65 phosphorylation during the time course, as observed by rise of the Lys63/pSer65 peptide (K63pS65).
AQUA analysis after 180 min as in (D) with Lys6‐, Lys11‐, Lys33‐, Lys48‐, Lys63‐, and Met1‐linked tetraUb (K6, K11, K33, K48, K63, M1). For all tetraUb chains, Ser65 is phosphorylated to greater than 80% after incubation with PhPINK1 for 180 min.
Time course analysis of autoubiquitination reactions with unphosphorylated full‐length human Parkin (hParkin) and Ub (left), a 80% Ub/20% phosphoUb mixture (middle) and 100% phosphoUb (right).
Analysis of different ratios of Ub:phosphoUb in a 120‐min ubiquitination reaction of full‐length hParkin. Parkin is activated by a mixture of Ub and phosphoUb, and is much less active in a reaction with no phosphoUb as well as with 100% phosphoUb.
Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and hydrolysis (2014)
Figure 2

NMR analysis of phosphoUb

A 1H, 15N‐BEST‐TROSY spectrum of phosphoUb (shades of blue) overlaid with wild‐type (wt) Ub (orange). The phosphoUb spectrum contains 130 non‐sidechain resonances that have been colored in dark blue for the major species and light blue for the minor species based on the assignment of each species in Supplementary Fig S2B–D.
B–D Weighted chemical shift perturbation (CSP) graphs for (B) wt Ub versus major phosphoUb species, (C) wt Ub versus minor phosphoUb species, (D) major versus minor phosphoUb species.
E Comparison of Ub secondary structure [derived from pdb‐id 1ubq (Vijay‐Kumar et al, )] with the secondary structure predictions of major and minor phosphoUb forms as calculated from backbone chemical shifts using TALOS+ (Supplementary Fig S5).
Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and hydrolysis (2014)
Figure 3

Crystal structure of Ser65‐phosphoUb

Structure of phosphoUb in space group P1 with 8 molecules in the asymmetric unit (Table ). Ub molecules are colored differently, and atoms for pSer65 are shown as spheres with red oxygen and purple phosphorous atoms.
Six of eight phosphoUb molecules form similar dimer interactions, where phosphoUb molecules interact via their Ile44 patch. This resembles the crystal structure of Lys48‐linked diUb [pdb‐id 1aar (Cook et al, )]. Atoms for pSer65 are shown as in (A), and the Ile44 side chain is shown as blue sticks.
Representative 2|Fo|‐|Fc| electron density, contoured at 1 σ, covering the phosphorylation site. pSer65 is shown in ball‐and‐stick representation, and neighboring side chains are shown in stick representation with red oxygen and blue nitrogen atoms.
In wt Ub (left), Ser65 forms a hydrogen bond with the backbone amide of Gln62. Upon phosphorylation (right), an oxygen atom from the phosphate forms the same backbone hydrogen bond.
Cartoon representation and electrostatic surface potential of Ub (left) and phosphoUb (right) in identical orientations. Electrostatics were calculated using CHARMM (www.charmm-gui.org) and are colored from red (negative potential) to blue (positive potential).
Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and hydrolysis (2014)
Figure 4

The minor conformation of phosphoUb

Long‐range HNCO experiments showing differences of hydrogen bonding patterns for major and minor phosphoUb species. Through H bond correlations (blue) recorded for residue Ile44 in the major and minor species of phosphoUb with reference to a control HNCO spectrum (red). In the major form of phosphoUb (left), the HN of Ile44 is a H bond donor to His68, as seen in wt Ub. In the minor form of phosphoUb (right), the HN of Ile44 does not have a cross peak to His68 but instead has a correlation to Val70.
Comparison of hydrogen bonding patterns for the β‐sheet portion of wt Ub [left, according to Nisius and Grzesiek ()], the major form of phosphoUb (middle) and the minor form of phosphoUb (right). Four Leu residues (green) undergo a two‐residue strand slippage to satisfy the observed hydrogen bonding pattern in the minor species. The green arrows indicate the resonances exemplarily shown in (A).
Structure of the major form of phosphoUb, where phosphoUb residues 1–61 are shown under a surface, and the β5 strand is shown in stick representation with red oxygen, blue nitrogen and purple phosphorus atoms. The four core Leu residues are shown in green.
Model of the phosphoUb minor species as in (C) in which the β5 strand is retracted into the protein core by two residues. This extends the β4–β5 loop and significantly shortens the C‐terminal tail of Ub.
Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and hydrolysis (2014)
Figure 5

Impact of Ub phosphorylation on E1 and E2 enzymes

PhosphoUb is abbreviated to pUb in this Figure.

E1‐mediated charging of E2 enzymes by Ub and phosphoUb in a time‐course analysis. Non‐reducing Coomassie‐stained 4–12% SDS–PAGE gradient gels are shown, and bands are labeled. “˜Ub” refers to generation of a thioester, while “Ub‐“refers to additional covalent ubiquitination of the E2 enzyme. * contaminant from E2 purification.
SAXS‐derived ensemble of UBE2D3 (Pruneda et al, ), where the E2 is shown under a blue surface and the Ub is shown as a green ribbon. Ser65 is shown in sphere representation. Ser65 does not contact the E2 enzyme in any orientation of the Ub at the E2 enzyme. PDB files were downloaded from the Klevit‐lab website (http://depts.washington.edu/klvtlab/).
Quantitative mass spectrometry analysis for UBE2T, UBE2E1 and UBE2R1 with Ub and phosphoUb using Ub AQUA peptides reveals impaired chain formation with phosphoUb (see also Supplementary Fig S10).
Charging of UBE2N, and UBE2N/UBE2V1‐mediated Lys63 chain assembly with Ub and phosphoUb. Charging of UBE2N as in (A) proceeds identically with both Ub and phosphoUb (left 6 lanes), while UBE2V1‐mediated Lys63 chain assembly only proceeds with Ub but not phosphoUb (right 6 lanes and Western blot).
Structure of Saccharomyces cerevisiae Ubc13‐Mms2 (blue/red, Mms2 is a homolog of UBE2V1) with the acceptor Ub (cyan) bound non‐covalently to Mms2 [pdb‐id 2gmi (Eddins et al, )]. Ser65 of the acceptor Ub interacts with Mms2, and Ser65 phosphorylation would prevent binding due to Mms2 Glu18.
PhosphoUb competition assays for UBE2N/UBE2V1‐mediated Lys63 diUb formation. Time course with K63R Ub (donor) and Ub ΔGG (acceptor) generates Lys63 diUb and is not inhibited upon increasing concentrations of phosphoUb, revealing phosphoUb does not compete with Ub for UBE2N/UBE2V1‐mediated Lys63 diUb formation.
Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and hydrolysis (2014)
Figure 6

E3‐mediated autoubiquitination with Ub and phosphoUb

A Time course for a ligase reaction with GST‐tagged cIAP1 (aa 363–614), UBE2D1 and Ub or phosphoUb. See Supplementary Fig S11D for a reaction with UBE2D3.
B Anti‐Ub Western blot of an identical reaction run for 3 h.
C Comparison of the number of Ub chain linkages in the reaction in (B).
D Ub chain linkage profile for the reaction in (B).
E Reaction of GST‐tagged TRAF6 (aa 1–285) with UBE2D3 as in (A). See Supplementary Fig S11E and F for additional TRAF6 reactions.
F Anti‐Ub Western blot of GST‐TRAF6/UBE2D1 run for 3 h.
G Comparison of the number of Ub chain linkages in the reaction in (F).
H Linkage composition in GST‐TRAF6‐UBE2D1 reactions reveals 58% Lys63 chains with wt Ub and UBE2D1. Significantly smaller quantities of linkages are assembled with phosphoUb.
I–K Chain assembly as in (A) with UBE2L3 and (I) GST‐tagged NEDD4L (aa 576–955) (Mund & Pelham, ), (J) NleL (aa 170–782) (Hospenthal et al, ) and (K) HOIP RBR‐LDD (aa 699–1072) (Smit et al, ). While the HECT E3 ligases NEDD4L and NleL are active with Ub and phosphoUb, HOIP is significantly less active with phosphoUb. See also Supplementary Fig S11G and H.
Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and hydrolysis (2014)
Figure 7

Binding of TAB2 NZF to phosphoUb chains

Structure of TAB2 NZF (red) in complex with Lys63 diUb (cyan) [pdb‐id 2wwz (Kulathu et al, )]. Ser65 for both Ub moieties is shown as spheres and highlighted with a blue circle.
Pull‐down assays with the Lys63‐specific TAB2 NZF against different tetraUb (Ub4) species are shown. Phosphorylation of K63 Ub4 does not prevent binding to TAB2 NZF. Met1‐linked tetraUb does not interact with TAB2 NZF, independent of Ser65 phosphorylation.
Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and hydrolysis (2014)
Figure 8

Defects in hydrolyzing phosphoUb chains

A Structure of USP2 (red) bound to Ub (cyan) [pdb‐id 2hd5 (Renatus et al, )], with a close‐up on Ub Ser65 in the distal Ub. A negative charge at the USP2 Asp345 position is conserved in most USP enzymes (Ye et al, ).
B–H Ubiquitination reactions from Fig A were used as input for deubiquitinase (DUB) analysis. Silver‐stained time‐course gels are shown. All DUBs tested (USP2, USP8, USP15, USP30, Ataxin‐3, USP21) hydrolyze Ub chains but have significantly lower activity against phosphoUb chains, with the exception of vOTU and USP21.
I USP30 activity was significantly lower for cIAP polyUb as compared to other USP DUBs. Cleavage assays against the diUb panel reveals preference for Lys6‐linked chains.
J USP30 activity is decreased against phosphorylated Lys6 diUb.
K–N Deubiquitinase assays against phosphorylated tetraUb. The activity of AMSH (K), TRABID (L), OTUB1 (M) and OTULIN (N) are inhibited with Lys63, Lys33, Lys48 and Met1 phosphorylated tetraUb, respectively.
YTHDF1-Mediated Translation Amplifies Wnt-Driven Intestinal Stemness (2020)
Figure 7

Figure 7. Targeting YTHDF1 in ISCs of established tumors leads to tumor shrinkage and prolonged survival

(A) Workflow of tamoxifen (TAM)-induced Ythdf1 deletion in AOM/DSS-induced Lgr5-creERT2:Ythdf1fl/fl mice.

(B) Colons from mice in (A) treated with or without TAM on day 80 of AOM/DSS induction.

(C) Colon tumor number from mice treated with or without TAM on day 80 of AOM/DSS induction. Data are shown as mean ± SEM (7 mice for each group). ****p<0. 0001 (t-test).

(D) Immunoblot analysis of 2 pairs of colon tumors from mice treated with or without TAM.

(E) Workflow of tamoxifen (TAM)-induced Ythdf1 deletion in Apcmin/+ mice.

(F) Kaplan-Meier survival plots for Apcmin/+; Lgr5-creERT2:Ythdf1fl/fl mice (8 mice for each group) treated with or without TAM. Log-rank (Mantel Cox) test. **p<0. 01 (t-test).

(G) A proposed model of amplified β-catenin activity by m6A-YTHDF1-mediated translation.

EXPANDED VIEW FIGURE LEGENDS
Transgenic mice with expanded neocortex due to human ARHGAP11B exhibit increased memory flexibility (2021)
Figure 1

Figure 1 - Increase in BP proliferation in 11B mouse neocortex at E14.5.

A Representative immunofluorescence for PH3 (green), combined with DAPI staining (white), of E14.5 wildtype (WT) and 11B mouse dorsolateral neocortex rostrally.

B Representative immunofluorescence for pVim (yellow), combined with DAPI staining (white), of E14.5 WT and 11B mouse dorsolateral neocortex rostrally. Boxes (50 µm x 65 µm) in (B) indicate areas that are shown at higher magnification in (B'). Arrow, pVim+ BP with basal process (mitotic bRG); notched arrowheads, basal process; arrowhead, pVim+ BP without basal process (mitotic bIP).

C-E Quantification of PH3+ APs (C), PH3+ BPs (D) and pVim+ bRG (E left) and bIPs (E right) in a 200 µm-wide field of E14.5 WT (white) and 11B (black) mouse dorsolateral neocortex rostrally. Note the difference in ordinate scales in (E).

F Representative immunofluorescence for Pax6 (cyan) and Tbr2 (magenta), combined with DAPI staining (white), of E14.5 WT and 11B mouse dorsolateral neocortex rostrally. Boxes (25 µm x 25 µm) in (F) indicate areas that are shown at higher magnification in (F'). Outlined by dashed white lines, Pax6+ & Tbr2+ BP; outlined by solid white lines, Pax6+ & Tbr2- BP.

G-K Quantification of Pax6+ & Tbr2- cells in VZ (G), Pax6+ & Tbr2+ cells in VZ (H), Pax6+ & Tbr2- cells in SVZ and IZ (I), Pax6+ & Tbr2+ cells in SVZ and IZ (J) and Pax6- & Tbr2+ cells in SVZ and IZ (K) in a 200 µm-wide field of E14.5 WT (white) and 11B (black) mouse dorsolateral neocortex rostrally.

L-N, Sequential BrdU-EdU labelling. BrdU was injected into pregnant mice at E13.5, i.e. 24 hr before sacrifice at E14.5, and EdU was injected into the same pregnant mice 0.5 hr before sacrifice at E14.5. (L) Representative immunofluorescence for BrdU (magenta), combined with EdU staining (cyan) and DAPI staining (white), of E14.5 WT and 11B mouse dorsolateral neocortex rostrally. (M, N) Quantification of BrdU+ & EdU+ cells (M) and the percentage of BrdU+ cells that are BrdU+ & EdU+ (N) in VZ and SVZ in a 200 µm-wide field of E14.5 WT (white) and 11B (black) mouse dorsolateral neocortex rostrally.

Data information: Images are single optical sections (A, B, B', L) and Z projections of 4 optical sections (F, F'). Scale bars, 50 µm (A, B, F, L), 10 µm (B', F'). Data are the mean of 6-19 (WT) and 6-26 (11B) littermate embryos, which were derived from 3-9 separate litters. Error bars indicate SD, two-tailed unpaired Student's t-test, * P < 0.05; ** P < 0.01 (C, D, E, G-K, M, N). P = 0.0269 (D); P = 0.0302 (E, left); P = 0.0139 (E, right); P = 0.0281 (I); P = 0.0088 (J); P = 0.0014 (K); P = 0.0049 (M, SVZ); P = 0.0323 (N, SVZ).
Transgenic mice with expanded neocortex due to human ARHGAP11B exhibit increased memory flexibility (2021)
Figure 2

Figure 2 - Neocortex expansion and increased cortical neuron production in embryonic 11B mouse neocortex at E18.5.

A Representative DAPI staining (white) of E18.5 wildtype (WT) and 11B mouse neocortex illustrating the measurements of cortical perimeter (blue), cortical thickness (red) and ventricular length (green).

B-G Quantification across the rostral-caudal axis (sections 1-9, B-D) and only the rostral part of the brain (sections 1-3, E-G) of cortical perimeter (B, E), cortical thickness (C, F) and ventricular length (D, G) of E18.5 WT (white) and 11B (black) mouse neocortex.

H Representative immunofluorescence for Tbr1 (cyan), Ctip2 (magenta) and Satb2 (green), combined with DAPI staining (white), of E18.5 WT and 11B mouse neocortex at the position where cortical thickness was measured (A, red lines).

I Quantification of zone thickness of E18.5 WT (white) and 11B (black) mouse neocortex at the position where cortical thickness was measured (A, red lines).

J-L Quantification of Tbr1+ (J), Ctip2+ (K) and Satb2+ (L) neurons in the CP in a 100 µm-wide field of E18.5 WT (white) and 11B (black) mouse neocortex at the position where cortical thickness was measured (A, red lines).

Data information: Images are single optical sections (A, H). Scale bar, 1 mm (A), 20 µm (H). Data are the mean of 7 (WT) and 8-12 (11B) littermate embryos, which were derived from 4 separate litters. Error bars indicate SD. Two-way ANOVA, followed by Bonferroni's multiple comparisons test (B-D); two-tailed unpaired Student's t-test (E-G, I-L), * P < 0.05, ** P < 0.01, **** P < 0.0001. WT vs. 11B, P < 0.0001; section number 3, P = 0.0322; section number 8, P = 0.0411 (B). WT vs. 11B, P < 0.0001; section number 1, 2, P = 0.0233; section number 3, P < 0.0001 (C). P = 0.0476 (E); P < 0.0001 (F); P = 0.0287 (I, IZ); P = 0.0039 (I, CP); P = 0.0085 (J); P = 0.0093 (K); P = 0.0157 (L).
Transgenic mice with expanded neocortex due to human ARHGAP11B exhibit increased memory flexibility (2021)
Figure 3

Figure 3 - Persistence of neocortex expansion and increased cortical neuron numbers in adult 11B mouse brain at P56.

A Dorsal view of adult wildtype (WT) and 11B mouse brain at P56. Orange lines illustrate the measurements of neocortex width and length; red lines illustrate the measurements of neocortex area.

B, C Quantification of neocortex width, length and area.

D Representative DAPI staining (white) of P56 adult WT and 11B mouse neocortex illustrating the measurements of cortical perimeter (blue) and thickness (red).

E, F Quantification of cortical perimeter (E) and thickness (F) in the rostral part of P56 adult WT and 11B mouse brain.

G, H Representative immunofluorescence for Ctip2 (magenta), Satb2 (green) and Brn2 (yellow), combined with DAPI staining (white), of P56 adult WT and 11B mouse neocortex at the position where cortical thickness was measured (D, red lines).

I, J Quantification of zone thickness (I) and layer thickness (J) of P56 adult WT and 11B mouse neocortex at the position where cortical thickness was measured (D, red lines).

K-M Quantification of Satb2+ (K), Brn2+ (L) and Ctip2+ (M) neurons in a 200 µm-wide field of P56 adult WT and 11B mouse neocortex.

Data information: Images are single optical sections (D, G). Scale bar, 1 mm (A, D), 20 µm (G). Data are the mean of 8 (WT) and 8 (11B) adult littermate mice. Error bars indicate SD, two-tailed unpaired Student's t-test (B, C, E, F, I-M), * P < 0.05, ** P < 0.01, **** P < 0.0001. P < 0.0001 (B, neocortex width); P < 0.0001 (C); P = 0.0297 (E); P = 0.0092 (F); P = 0.0227 (I, CP); P = 0.0308 (J, layer II+III); P = 0.0278 (K); P = 0.0133 (L).
Loss of Nat4 and its associated histone H4 N-terminal acetylation mediates calorie restriction-induced longevity (2016)
Figure 1

Figure 1. NAT4 deletion extends lifespan through a calorie restriction-mediated pathway.

(A) Expression levels of NAT4 analyzed by qRT-PCR using total RNA extracted from a wild-type (BY4741) and Pste5-NAT4 strain grown in 2% (NCR), or 0.1% glucose (CR). NAT4 levels were normalized to TAF10, whose expression remains unchanged. Error bars, SEM (Standard Error of the Mean) of 3 independent experiments. ** p ≤ 0.01; calculated by unpaired two-tailed Student’s t-test. (B) ChIP analysis performed in BY4741 wild-type, nat4∆ and Pste5-NAT4 strains grown in the same conditions as in (A). Chromatin was immunoprecipitated using an antibody against N-acH4 and analyzed by qRT-PCR using primers for the different rDNA regions. Enrichment was normalized to Histone H4 levels. Error bars, SEM of 3 independent experiments. Statistical significance was determined by unpaired two-tailed Student’s t test: * p ≤ 0.05; ** p ≤ 0.01 compared values to wild-type NCR conditions; arrowhead p ≤ 0.05 compared values to wild-type CR conditions (C) Replicative lifespan (RLS) for BY4741 wild-type and nat4Δ strains. Values in parenthesis (here and hereafter) indicate mean lifespan. Statistical significance (here and hereafter) was determined by one-way ANOVA test: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. (D) Mean RLS for wild-type and nat4∆ strains using different genetic backgrounds. Values outside of parenthesis indicate mean lifespan, while values in parenthesis indicate the number of cells examined. (E) Mean RLS for BY4741 wild-type and nat4∆ strains under NCR and CR conditions. (F) Mean RLS for BY4741 wild-type and Pste5-NAT4 strains under NCR and CR conditions.
Loss of Nat4 and its associated histone H4 N-terminal acetylation mediates calorie restriction-induced longevity (2016)
Figure 2

Figure 2. rDNA stability and mRNA translation are unaffected in nat4∆.

(A) Schematic of the budding yeast rDNA locus on chromosome XII. Primers along the rDNA locus are indicated by red lines and numbers 1 to 10. NTS (Non Transcribed Spacer), ETS (External Transcribed Spacer), ITS (Internal Transcribed Spacer), RFB (Replication Fork Barrier), E-pro (Bidirectional promoter) (B) ChIP experiments were performed in SF10 wild-type, nat4∆, NET1-TAP and NET1-TAP nat4∆ strains using an anti-IgG antibody. The enrichment from the antibody was normalized to input. Error bars, SEM of 3 independent experiments. Statistical significance was determined by unpaired two-tailed Student’s t test: ** p ≤ 0.01 compared nat4∆ to WT values. (C) Quantitative real-time PCR analysis of rDNA copy number for young and old cells in BY4741 wild-type, nat4∆ and fob1∆ strains. Error bars, SEM of 3 independent experiments. Statistical significance was determined by unpaired two-tailed Student’s t test: ** p ≤ 0.01 compared to corresponding WT values. (D) Polysome profiling analysis performed in BY4741 wild-type and nat4∆ strains. The plots are representative of two independent experiments.