EMBO/soda-nel huggingface dataset:
MAPK Signaling Regulates c-MYC for Melanoma Cell Adaptation to Asparagine Restriction (2020)
Figure 4

Figure 4. c-MYC Regulates Essential Amino Acid Uptake and mTORC1 Activity to Support AAR Signaling.

A. Immunoblotting of indicated proteins in A375 cells 72 hr after treatment with siASNS, siSLC1A5, siSLC7A5, or a combination of siASNS and siSLC1A5, or siASNS and siSLC7A5.

B. RT-qPCR analysis of indicated transcripts in melanoma cells after 36 hr treatment with siASNS, siSLC7A5, or both.

C. Proliferation of melanoma cells over indicated times after treatment with siASNS, siSLC7A5, or both.

D. GC-MS-based estimation of intracellular amino acid levels in A375 cells treated with siASNS, siATF4, or both (top left); siASNS, sicMYC, or both (top right); siASNS, siSLC1A5, or both (bottom left); or siASNS, siSLC7A5, or both (bottom right). The fold change is relative to NT-siRNA-treated A375 cells.

E-K. Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment (E), treatment with siASNS, sicMYC, or both (F), siASNS, siSLC7A5, or both (G), siASNS, BCH, or both (H), siASNS, HA-GSK3-β (S9A), or both (I) , siASNS or indicated combinations (J), or siASNS, siSLC3A2, or both (K).

L. RT-qPCR analysis of indicated transcripts in melanoma cells after 36 hr treatment with siASNS, siSLC3A2, or both.

M. Proliferation of melanoma cells over indicated times after treatment with siASNS, siSLC3A2, or both.

Data information: Data are shown as the mean ± SD, n = 3 biological replicates. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Unpaired Student's t-test was used in (B), (D), and (L); two-way ANOVA was utilized in (C) and (M). In (D), the statistical significance denoted in black and orange corresponds to the comparison of NT-siRNA vs. siASNS and siASNS vs. siATF4, sicMYC, siSLC1A5, or siSLC7A5, respectively. Statistical significance shown only for the combination treatments reversing the effect of siASNS on the intracellular amino acid levels. In (D), siATF4 and siSLC7A5 graphs are part of the same experiment and sicMYC and siSLC1A5 graphs are part of the same experiment. The intracellular levels of histidine and tryptophan couldn't be measured in siASNS+siATF4 and siASNS+siSLC7A5 combination settings, and, therefore, not included shown. The statistical significance denoted in black and orange corresponds to the comparison of siASNS+siSLC7A5 vs. siASNS and siSLC7A5 respectively (C) and the comparison of siASNS+siSLC3A2 vs. siASNS and siSLC3A2 respectively (M).
Tryptophan-rich basic protein (WRB) mediates insertion of the tail-anchored protein otoferlin and is required for hair cell exocytosis and hearing (2016)
Figure 2

Figure 2. The TRC40 pathway mediates the insertion of otoferlin into mammalian ER-derived microsomes

A. HZZ-OTOFop, carrying a C-terminal glycosylation site (opsin tag) was purified alone or in complex with wild-type or an ATPase-deficient mutant version of TRC40 and incubated in the absence or presence of ER-derived rough microsomes (RM). Membrane integration (glycosylation) was monitored by SDS-PAGE and immunoblot using an anti-opsin antibody. Where indicated, EndoH was used to remove N-linked oligosaccharides.

B. HZZ-OTOFop in complex with wild-type or mutant TRC40 was incubated with RM in the presence or absence of ATP and membrane insertion was monitored by opsin-specific immunoblot.

C. HZZ-OTOFop in complex with wild-type TRC40 was incubated in the presence of RM, trypsin-treated rough microsomes (TRM) or in the presence of WRBcc or CAMLcyt. Membrane insertion was monitored by opsin-specific immunoblot.

D. Quantification of relative protein glycosylation shown in B (n = 3). Data are represented as mean ± SEM. TRC40gr, mutated version of TRC40; **p<0.01; ***p<0.001 (Student’s two-sample t-test).

E. Quantification of relative protein glycosylation of the data shown in C (n = 3). Data are represented as mean ± SEM. ***p<0.001 (Student’s two-sample t-test)
Tryptophan-rich basic protein (WRB) mediates insertion of the tail-anchored protein otoferlin and is required for hair cell exocytosis and hearing (2016)
Figure 3

Figure 3. Wrb disruption in IHCs causes a progressive hearing impairment in mice

A. Grand averages of auditory brainstem responses (ABR) from Wrbfl/fl:CreA (red traces; SEM pink) and Wrb+/+:CreA (black traces; SEM grey) mice. ABRs were recorded for three separate age groups (A-A’’, as indicated in the graph) using click stimulation at 80 dB (peak equivalent) and 20 Hz stimulation rate. There was an age- progressive reduction of ABR amplitude in Wrbfl/fl:CreA mice.

B. ABRs from Wrbfl/fl:CreA mice showed a progressive threshold increase, Wrb+/+:CreA mice and Wrbfl/fl mice lacking Cre recombinase had normal thresholds. Statistical comparison of the threshold at 12 kHz was done by Kruskal-Wallis test, p values are from post-hoc Dunn’s multiple comparison test. Measurements in which no ABR was observed at the maximal available tone burst level (90 dB) or click level (120 dB) scored as 100 dB threshold. Data are represented as mean ± SEM.
Tryptophan-rich basic protein (WRB) mediates insertion of the tail-anchored protein otoferlin and is required for hair cell exocytosis and hearing (2016)
Figure 4

Figure 4. Wrb disruption in mouse IHCs greatly reduced otoferlin levels

A-D. Representative maximum projections of confocal sections of (A-B) P14 and (C-D) P20 Wrb+/+:CreA and Wrbfl/fl:CreA apical turn organs of Corti following immunolabeling against otoferlin (green) and Vglut3 (magenta) processed and imaged under identical conditions. A’-D’ show individual otoferlin stainings with an intensity-coded lookup table. A’’-D’’ present single xz projections at a central point through a representative IHC to illustrate otoferlin subcellular distribution. Scale bars in A-D’ represent 10 μm, in A’’-D’’ 2 µm. Note the dramatic reduction in otoferlin fluorescence and the alteration in its distribution as well as a change in cell shape and position of nuclei (for detailed analysis of cell shape and nuclei position, please refer to Figure EV5).

E-G. Semiquantitative analysis of otoferlin immunofluorescence in the apical parts of IHCs from Wrbfl/fl:CreA mice using averaged regions of interests in cell’s maximal projections (outlined with a dotted line in G for a single representative IHC) normalized to the maximum values observed in the respective control IHCs. Averaged supranuclear coronal line profiles as illustrated with a dashed line in G are shown for (F) P14-16 and (F’) P18-21 IHCs. ***p<0.001 versus Wrb+/+:CreA

H-J. Semiquantitative analysis of otoferlin immunofluorescence in the basal parts of IHCs from Wrbfl/fl:CreA mice using averaged regions of interests in cell’s maximal projections (outlined with a dotted line in J for a single representative IHC) normalized to the maximum values observed in the respective control IHCs. Averaged supranuclear coronal line profiles as illustrated with a dashed line in G are shown for (I) P14-16 and (I’) P18-21 IHCs. ***p<0.001 versus Wrb+/+:CreA

All data are represented as grand averages of individual cell means ± SEM normalized to their respective Wrb+/+:CreA control.
Tryptophan-rich basic protein (WRB) mediates insertion of the tail-anchored protein otoferlin and is required for hair cell exocytosis and hearing (2016)
Figure 5

Figure 5. Fewer ribbon-associated synaptic vesicles and perisynaptic accumulation of cisternal organelles and rough ER in Wrb-deficient IHCs

A. Representative electron micrograph of a Wrb+/+:CreA IHC ribbon synapse. Scale bar: 120 nm.

B-B’. In Wrbfl/fl:CreA (depicted are two consecutive ultrathin sections) IHCs, accumulations of large partially amorphous vesicles were observed close to ribbon synapses (red arrows), but also further away in the cytoplasm (cyan arrows). Moreover, cisternal structures resembling (r)ER are found unusually close to the ribbon (orange arrow in B’). Scale bar: 120 nm.

C. 3D serial reconstruction of the ribbon synapse depicted in (B, B’, using “Reconstruct” software). Scale bar: 100 nm.

D. Schematic illustration of the quantitative analysis performed on random ultrathin sections, results shown in (E) and (F).

E. Quantification in random ultrathin sections (Wrb+/+:CreA n = 28, from two animals; Wrbfl/fl:CreA n = 44, from three animals) of the numbers of membrane-proximal “MP” vesicles (in close proximity to membrane and ribbon); vesicles within a 100 nm range along the active zone membrane; vesicles at the lower “proximal” and the upper “distal” half of the ribbon as well as vesicle >70 nm (“large vesicles”) in a radius of 800 nm around the ribbon. The vesicle number was significantly reduced in Wrbfl/fl:CreA IHCs at both ribbon parts but not at the presynaptic membrane.

F. Average vesicle diameters of the membrane-proximal vesicles and the vesicles at both halves of the ribbon. The mean vesicle diameter increases significantly at both parts of the ribbon in Wrbfl/fl:CreA IHCs. Data are represented as mean ± SEM. Student’s two-sample t-test ***p<0.001
Tryptophan-rich basic protein (WRB) mediates insertion of the tail-anchored protein otoferlin and is required for hair cell exocytosis and hearing (2016)
Figure 6

Figure 6. Impaired sustained exocytosis in Wrb-deficient IHCs

A. Ca2+ current-voltage relationship of P14-P17 IHCs of Wrbfl/fl:CreA (n = 17 IHCs) and Wrb+/+:CreA (n = 15 IHCs) mice in 2 mM extracellular [Ca2+] showed no change in amplitude or voltage-dependence of the Ca2+ current. Data are represented as mean ± SEM.

B. Representative Ca2+ current (top) and Cm changes (bottom) in Wrbfl/fl:CreA and Wrb+/+:CreA IHCs in response to a 20-ms step depolarizations to -14 mV (the potential eliciting the maximum Ca2+ current).

C. Representative Ca2+ current (top) and Cm changes (bottom; finite impulse response filtered) in Wrbfl/fl:CreA and Wrb+/+:CreA IHCs in response to a 100 ms step depolarizations to -14 mV.

D. Exocytic ΔCm (top) and corresponding Ca2+ current integrals, QCa (bottom) for various depolarization durations. Data are grand averages of the cells’ means. Exocytic ΔCm was reduced in the knockout from 20 ms onwards, indicating reduced sustained exocytosis. Data from OtofPga/Pga animals were replotted for direct comparison from (Pangrsic et al, 2010).

E. ΔCm/QCa2+ ratio indicates a lower efficacy of Ca2+ influx in driving exocytosis in Wrbfl/fl:CreA IHCs, for stimuli of 20 ms or longer. Data represents means ± SEM; *p<0.05; **p<0.01; ***p<0.001
Tryptophan-rich basic protein (WRB) mediates insertion of the tail-anchored protein otoferlin and is required for hair cell exocytosis and hearing (2016)
Figure 7

Figure 7. Impaired sound encoding at afferent IHC synapses

A. All SGNs of Wrbfl/fl:CreA mice had spontaneous firing rates (SR) below 20 Hz (p>0.05, Kolmogorow-Smirnov test).

B. Representative tuning curves from Wrbfl/fl:CreA and Wrb+/+:CreA SGNs indicating preserved active cochlear amplification despite disruption of Wrb in IHCs.

C. Normal thresholds at the characteristic frequency (CF) in Wrbfl/fl:CreA SGNs.

D-D’’. Mean poststimulus time histograms (PSTH) ± SEM of Wrbfl/fl:CreA and Wrb+/+:CreA SGNs in response to 50 ms tone bursts presented at CF, 30 dB above threshold at stimulus rates of 2 Hz (D), 5 Hz (D’), or 10 Hz (D’’). While the general response pattern was preserved, spike rates were drastically reduced in Wrbfl/fl:CreA SGNs, especially at higher stimulus rates. Data from auditory neurons (SGN and cochlear nucleus) of OtofPga/Pga animals were replotted for direct comparison from (Pangrsic et al, 2010).

E. Spike rates in response to sound onset (maximum rate in PSTH with 0.5 ms binwidth) and adapted rates (averaged between 35-45 ms after stimulus onset) were significantly lower in Wrbfl/fl:CreA SGNs (stimulus rate 5 Hz; p<0.001).

F. In line with the reduction in spike rates, first spike latency (FSL) following stimulus onset was greatly increased in Wrbfl/fl:CreA SGNs compared to Wrb+/+:CreA (stimulus rate 5 Hz; p<0.001).

G. Illustration of the stimulus paradigm for forward masking experiments: a 100 ms masker tone presented at CF, 30 dB above threshold was followed by a silent interval of variable duration and a 15 ms probe tone (CF, 30 dB above threshold). Bottom: in Wrbfl/fl:CreA SGNs, the response to the first 5 ms of the masker probe tone tone (shown as a fraction of the response to the first 5ms of the masker) was strongly reduced, and the half time of recovery (from normalized curves) was increased from 39.2 ± 12.2 ms in Wrb+/+:CreA to 215.7 ± 51.2 ms in Wrbfl/fl:CreA SGNs (p<0.001, Mann-Whitney U test). Data represent means ± SEM.
Tryptophan-rich basic protein (WRB) mediates insertion of the tail-anchored protein otoferlin and is required for hair cell exocytosis and hearing (2016)
Figure 8

Figure 8. Isoleucin deletion in the transmembrane domain of otoferlin causes impaired ER-targeting in vitro and hearing impairment in a human patient

A. Electropherograms of the sequences containing the mutations observed in family E952. Position within the OTOF cDNA (NM_001287489.1) is indicated above each sequence. For each mutation, the wild-type sequence and the heterozygous sequence are shown. For purposes of clarity, the two alleles carried by subject E952-II:1 were subcloned into a plasmid vector and sequenced in separation. Sequences of the subcloned mutant alleles are shown in the bottom row.

B. Pedigree of family E952 showing the segregation of mutations in the OTOF gene.

C. Apparent decrease in insertion efficiency for the TMD mutant version. While the WT otoferlin glycosylation amounted to 3.84 ± 0.53, the TMD mutant had only 2.13 ± 0.65 (n=3, p<0.05). Data are represented as mean ± SEM.
The metabolite -ketoglutarate extends lifespan by inhibiting ATP synthase and TOR (2014)
f1

a, &agr;-KG extends the lifespan of adult worms in the metabolite longevity screen. All metabolites were given at a concentration of 8 mM. b, Structure of &agr;-KG. c, Dose–response curve of the &agr;-KG effect on longevity. d, e, &agr;-KG extends the lifespan of worms fed bacteria that have been ampicillin arrested, mean lifespan (days of adulthood) with vehicle treatment (mveh) = 19.4 (n = 80 animals tested), m&agr;-KG = 25.1 (n = 91), P  0.0001 (log-rank test) (d); or &ggr;-irradiation-killed, mveh = 19.0 (n = 88), m&agr;-KG = 23.0 (n = 46), P  0.0001 (log-rank test) (e). OP50, E. coli OP50 strain. f, &agr;-KG does not further extend the lifespan of ogdh-1 RNAi worms, mveh = 21.2 (n = 98), m&agr;-KG = 21.1 (n = 100), P = 0.65 (log-rank test).
The metabolite -ketoglutarate extends lifespan by inhibiting ATP synthase and TOR (2014)
f2

a, DARTS identifies ATP5B as an &agr;-KG-binding protein. Red arrowhead, protected band. b, DARTS confirms &agr;-KG binding specifically to ATP5B. IB, immunoblot. c, Inhibition of ATP synthase by &agr;-KG (released from octyl &agr;-KG; Supplementary Notes). This inhibition was reversible (data not shown). d, e, Reduced ATP levels in octyl &agr;-KG-treated normal human fibroblasts (**P = 0.0016, ****P  0.0001; by t-test, two-tailed, two-sample unequal variance) (d) and &agr;-KG-treated worms (day 2, P = 0.969; day 8, *P = 0.012; by t-test, two-tailed, two-sample unequal variance) (e). RLU, relative luminescence units. f, g, Decreased oxygen consumption rate (OCR) in octyl &agr;-KG-treated cells (***P = 0.0004, ****P  0.0001; by t-test, two-tailed, two-sample unequal variance) (f) and &agr;-KG-treated worms (P  0.0001; by t-test, two-tailed, two-sample unequal variance) (g). h, &agr;-KG, released from octyl &agr;-KG (800 µM), decreases state 3, but not state 4o or 3u (P = 0.997), respiration in mitochondria isolated from mouse liver. The respiratory control ratio is decreased in the octyl &agr;-KG- (3.1 ± 0.6) versus vehicle-treated mitochondria (5.2 ± 1.0) (*P = 0.015; by t-test, two-tailed, two-sample unequal variance). Oligo, oligomycin; FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; AA, antimycin A. i, Eadie–Hofstee plot of steady-state inhibition kinetics of ATP synthase by &agr;-KG (produced by in situ hydrolysis of octyl &agr;-KG). [S] is the substrate (ADP) concentration, and V is the initial velocity of ATP synthesis in the presence of 200 µM octanol (vehicle control) or octyl &agr;-KG. &agr;-KG (produced from octyl &agr;-KG) decreases the apparent Vmax (53.9 to 26.7) and Km (25.9 to 15.4), by nonlinear regression least-squares fit. c–i, Results were replicated in two independent experiments. Mean ± standard deviation (s.d.) is plotted in all cases.
The metabolite -ketoglutarate extends lifespan by inhibiting ATP synthase and TOR (2014)
f3

a–g, Effect of &agr;-KG on the lifespan of mutant or RNAi worms. a, atp-2 RNAi, mveh = 22.8 (n = 97), m&agr;-KG = 22.5 (n = 94), P = 0.35; or RNAi control, mveh = 18.6 (n = 94), m&agr;-KG = 23.4 (n = 91), P  0.0001. b, daf-2(e1370), mveh = 38.0 (n = 72), m&agr;-KG = 47.6 (n = 69), P  0.0001. c, eat-2(ad1116), mveh = 22.8 (n = 59), m&agr;-KG = 22.9 (n = 40), P = 0.79. d, let-363 RNAi, mveh = 25.1 (n = 96), m&agr;-KG = 25.7 (n = 74), P = 0.95; or gfp RNAi control, mveh = 20.2 (n = 99), m&agr;-KG = 27.7 (n = 81), P  0.0001. e, daf-16(mu86), mveh = 13.4 (n = 71), m&agr;-KG = 17.4 (n = 72), P  0.0001; or N2, mveh = 13.2 (n = 100), m&agr;-KG = 22.3 (n = 104), P  0.0001. f, pha-4(zu225), mveh = 14.2 (n = 94), m&agr;-KG = 13.5 (n = 109), P = 0.55. g, hif-1(ia4), mveh = 20.5 (n = 85), m&agr;-KG = 26.0 (n = 71), P  0.0001; or N2, mveh = 21.5 (n = 101), m&agr;-KG = 24.6 (n = 102), P  0.0001. P values were determined by the log-rank test. Number of independent experiments: RNAi control (6), atp-2 (2), let-363 (3), N2 (5), daf-2 (2), eat-2 (2), pha-4 (2), daf-16 (2), hif-1 (5).
The metabolite -ketoglutarate extends lifespan by inhibiting ATP synthase and TOR (2014)
f4

a, Decreased phosphorylation of mammalian TOR substrates in U87 cells treated with octyl &agr;-KG or oligomycin. Similar results were obtained in HEK-293 cells, normal human fibroblasts and mouse embryonic fibroblasts (data not shown). P, phospho. b, Increased autophagy in animals treated with &agr;-KG or RNAi for atp-2 or let-363. Photographs were taken at ×100 magnification. c, GFP::LGG-1 puncta quantified using ImageJ (Methods). Data show results of 2–3 independent experiments. Bars indicate the mean. ****P  0.0001; NS, not significant (t-test, two-tailed, two-sample unequal variance). d, &agr;-KG levels are increased in starved worms. **P  0.01 (t-test, two-tailed, two-sample unequal variance). Mean ± s.d. is plotted. e, Model of &agr;-KG-mediated longevity.
Biofabricating murine and human myo-substitutes for rapid volumetric muscle loss restoration (2021)
Figure 5

Figure 5. Immunofluorescence analysis on ablated TA muscle sections. Cross and longitudinal sections of ablated (control) and implanted (graft) TA were marked with LacZ and immunoassayed for MHC (red), LAM (green). Cross- and longitudinal sections were immunostained against MHC and LAM and overlaid to LacZ labeled images. Dashed area indicates the enlarged view displaying sarcomeres (asterisks) in combination with LacZ positive nuclei (arrowheads).
Biofabricating murine and human myo-substitutes for rapid volumetric muscle loss restoration (2021)
Figure 7

Figure 7. Innervation assessment on reconstructed TA sections. A) Immunofluorescence against NeuF (green) and MHC (red) revealing reconstructed muscle innervation (asterisks). B) Superimposed image in (a) over LacZ stained image. C) Syn and BTX immunolabelled sections showing neuromuscular junction formation (arrowheads), inset enlarged view of dashed box. D) Electrophysiological analysis scoring muscle strength recovery performed in vivo on ablated TA (control) and wet-spun implanted contralateral TA (graft) (n=3), statistical significance was analyzed by Anova test (P < 0.05 was considered significant: *55Hz=0,016830182; *75Hz=0,012686264; *100Hz=0,009143348; *150Hz=0,009327048). E) Rate of coupled versus uncoupled BTX-Syn signals in reconstructed TA NMJ.
Biofabricating murine and human myo-substitutes for rapid volumetric muscle loss restoration (2021)
Figure 8

Figure 8. Fabrication of human myoblast based myo-substitute. Wet-spun yarn containing human myogenic precursors showed progressive cell differentiation over time (arrows in day 3); dashed box enlargement reveals still undifferentiated cells after 15 days of culture (Upper panel). Middle and lower panel showing MHC immunostaining (red) performed on microfiber yarns and bulk structure loaded with human myoblasts upon 15 days of culture, revealing a significant difference in terms of myotube organization between the two hydrogel systems, with the wet-spun constructs greatly outperforming the bulk gels. Such difference is quantified in the polar plots. Nuclei were counterstained by DAPI (Blue).

Rat

TA

Gel injected in situ

multiple times

Cell sheets supported by

Bone-tendon anchors

Collagen type I

nanofibrillar strips

Mouse

Table 1. State-of-the-art of TA-VML reconstruction in small size animals (mice and rats).

Expanded View
TORC1 and TORC2 converge to regulate the SAGA co-activator in response to nutrient availability (2017)
Figure 1

Figure 1. SAGA is epistatic to the TORC1 and TORC2 pathways in the regulation of differentiation in response to nutrient availability. (A,B,D,E) Expression of ste11+ (A,D) and mei2+ (B,E) using quantitative RT-PCR of RNA extracted from cells grown either in nutrient rich medium (dark gray) or shifted for 4 hours to starvation medium (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), gcn5Η, tsc1Η, gcn5Η tsc1Η, tsc2Η, gcn5Η tsc2Η, rhb1-DA4 - a constitutively active (CA) rhb1 mutant [34], gcn5Η rhb1-DA4, tor2-L1310P - a CA tor2 mutant [33], gcn5Η tor2-L1310P, tor1Η, gcn5Η tor1Η, gad8Η, and gcn5Η gad8Η. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 4 (A,B) or 3 (D,E) independent experiments, overlaid with individual data points and standard error (SE) bars. Statistical significance was determined by 2-way ANOVA followed by Bonferroni"s multiple comparison tests (n = 4 for A,B) (n = 3 for D,E). (C,F) Cells were grown to mid-log phase either in rich medium or shifted for 8 hours to starvation medium. Zygotes and tetrads, which correspond to differentiated cells, were counted under a light microscope. Cells of the following genotypes were analyzed: WT isogenic controls, gcn5Η, tsc1Η, gcn5Η tsc1Η, rhb1-DA4, gcn5Η rhb1-DA4, gad8Η, and gcn5Η gad8Η. Each value represents the mean percentage and SE of differentiating cells to the total number of cells, averaged from 4 independent experiments. At least 200 cells from the indicated genotypes were counted in each experiment. White arrowheads indicate zygotes. Scale bar, 10 μm.
TORC1 and TORC2 converge to regulate the SAGA co-activator in response to nutrient availability (2017)
Figure 2

Figure 2. SAGA is phosphorylated in response to nutrient starvation. (A,B) 4-20% gradient SDS-polyacrylamide gel electrophoresis analysis of SAGA purified from cells grown either in rich medium (R) or starved for 45 minutes (S). SAGA was purified using endogenously TAP-tagged Ada1. A fraction of each eluate was loaded and either stained with silver, to detect all proteins (A), or with Pro-Q® Diamond, which stains phosphorylated proteins (B). A strain without any TAP tag is shown as a negative control for the purification. The graph on the left of the gel in B shows the fluorescence intensity of the phospho-specific stain, which was quantified along the left lane in blue (rich) and the middle lane in red (starved), using ImageJ. The area of one peak, which corresponds to the bands marked with arrowheads and was coined p55, is 1.8-fold larger in SAGA purified from starved cells, as compared to rich conditions. Below is an anti-HA immuno-blot (IB) of each eluate, to reveal the amount of Ada1-HA bait recovered. Shown are gels that are representative of 3 independent experiments. A.U., arbitrary units.