EMBO/soda-nel huggingface dataset:
Liver ASK1 protects from non-alcoholic fatty liver disease and fibrosis (2019)
Figure 4

Figure 4 Autophagy is impaired in hepatocyte-specific ASK1-knockout mice

(A) Shown is one representative Western blots from two independent experiments and quantification of respective protein levels in mice fed a HFD for 20 weeks (ASK1F/F n=8 mice; ASK1Δhep n=8 mice). (B) Representative immunofluorescence images of liver sections stained for LC3-II punctate (red) and nuclei (DAPI, blue) in mice. Scale bar represents 100 µm. Values are expressed as mean ± SEM. **p<0.01; ***p<0.001. Statistical tests used: t-test.
Liver ASK1 protects from non-alcoholic fatty liver disease and fibrosis (2019)
Figure 5

Figure 5 Depletion of ASK1 impairs autophagy in primary hepatocytes

(A) Primary hepatocytes isolated from chow-fed ASK1F/F () and ASK1Δhep () mice were treated with BSA, Palm, Palm+Rap or Palm+Rap+Baf for 24 h. Cells were stained for lipid droplet accumulation (Bodipy 493/503, green) and nuclei (Hoechst, blue). Lipid accumulation was quantified using automated image-based analysis (n=4 biological replicates). (B) Colocalization of LC3-II punctate (red) with lipid (BODIPY 493/503, green) and nuclei (Hoechst, blue) in primary hepatocytes treated with BSA, Palm, Palm+Rap or Palm+Rap+Baf. Scale bar represents 100 µm. Values are expressed as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001. Statistical tests used: ANOVA.
Liver ASK1 protects from non-alcoholic fatty liver disease and fibrosis (2019)
Figure 6

Figure 6 Pharmacological inhibition of ASK1 induces hepatic lipid accumulation

(A) Western blots and quantification of respective protein levels in liver of HFD-fed mice treated with or without ASK1 inhibitor (n=4 mice per treatment). (B) Representative images of liver sections stained with H&E (scale bar represents 100 µm) and (C) liver triglyceride (TG) content of HFD-fed mice treated with or without ASK1 inhibitor (n=8 mice per treatment). Values are expressed as mean ± SEM. *p<0.05; **p<0.01. Statistical tests used: t-test for A and C.
Liver ASK1 protects from non-alcoholic fatty liver disease and fibrosis (2019)
Figure 7

Figure 7 Liver-specific ASK1 overexpression ameliorates HFD-induced steatosis and protects from CCl4-induced fibrosis

(A) Representative images of liver sections stained with H&E from mice fed a HFD for 20 weeks. Scale bar represents 100 µm. (B) Liver triglyceride (TG) content (ASK1F/F n=11 mice; ASK1+hep n=10 mice) in livers of mice fed a HFD for 20 weeks. (C) Western blots and quantification of protein levels of respective targets in liver of HFD-fed mice (n=8 mice per genotype for pJNK and ATG12, n=4 mice per genotype for pBCN1). (D) Quantification of Sirius Red positive area (ASK1F/F n=11 mice; ASK1+hep n=9 mice) in livers of CCl4-injected mice. (E and F) Relative mRNA expression (ASK1F/F n=13 mice; ASK1+hep n=14 mice) of respective genes involved in inflammation and fibrosis in liver of CCl4-injected mice. Values are expressed as mean ± SEM. #p<0.1; *p<0.05; **p<0.01. Statistical tests used: t-test for B, C, D, E, F.
AKT isoforms modulate Th1-like Treg generation and function in human autoimmune disease (2016)
Figure 1

Figure 1. The PI3K/AKT/Foxo pathway is differentially expressed in Th1-Tregs

a. Fold change in mRNA expression of members of the PI3K/AKT/Foxo pathway in IFN+ as compared to IFN— Tregs in the microarray analysis. b. mRNA expression of members of the PI3K/AKT/Foxo signaling pathway in sorted IFN+ and IFN— Tregs. c. Sorted CD4+CD25highCD127— Tregs were stimulated in the presence of IL-2 (white) or IL-2+IL-12 (black dots) and RNA was isolated every 24 hours for 4 days. mRNA expression of members of the PI3K/AKT/Foxo signaling pathway. d. Representative example of the protein expression of AKT1, AKT2, AKT3, PTEN and FOXO3 on sorted CD4+CD25highCD127— Tregs stimulated in the presence of IL-2 (grey line) or IL-2+IL-12 (black line) and day 2 for AKT1, AKT2, AKT3 and PTEN and day 3 for FOXO3. Grey histogram represents the isotype. e. Statistical analysis of AKT1, AKT2, AKT3, FOXO3 and PTEN gMFI of n=6 experiments performed. * p<0.05, ** p<0.005, *** p<0.0005.
AKT isoforms modulate Th1-like Treg generation and function in human autoimmune disease (2016)
Figure 2

Figure 2. The PI3K/AKT/Foxo pathway is activated during Th1-Treg generation

Sorted CD4+CD25hiCD127— Tregs were stimulated with anti-CD3, anti-CD28 and IL-2 in the presence or absence of IL-12. a. Tregs were stimulated for 50 minutes with IL-12 or vehicle. Histograms show a representative example of n=8 independent experiments performed of the expression of phosphorylated molecules at 30 minutes after stimulation for pAKT and pFoxo1, and at 40 minutes for pFoxo3 in vehicle- (grey open histogram) or IL-12-treated (dark open histogram) Tregs. Grey histograms represent the isotype. The time points selected were based on kinetic experiments performed for a total of 50 minutes b. Tregs were stimulated in the presence (black) or absence (grey) of IL-12 and the expression of phosphorylated molecules was examined 16 hours later. c. gMFI of pAKT, pFoxo1 and pFoxo3 of n=6 independent experiments performed. d. Foxo1 and Foxo3 transcriptional targets expression on sorted CD4+CD25hiCD127— Tregs stimulated in the presence of vehicle or IL-12 at day 2 after stimulation (n=8). *p < 0.05, **p < 0.005, ***p < 0.0005.
AKT isoforms modulate Th1-like Treg generation and function in human autoimmune disease (2016)
Figure 3

Figure 3. Activation of the PI3K/AKT/Foxo pathway is sufficient to induce the Th1 phenotype on Tregs

a. Sorted CD4+CD25hiCD127— Tregs were stimulated in the presence of vehicle (white dots) or either SF1670 (first row), SC-79 (second row), 740 Y-P (third row) or AS1842856 (fourth row, black dots) for 3 days and IFNG and TBX21 gene expression was examined every 24 hours. Statistical analysis of n=4 independent experiments performed. b. Representative example of the frequency of IFN+Foxp3+ cells in Tregs stimulated in the presence of vehicle, SF1670, SC-79, 740 Y-P or AS1842856 for 3 days after 4 hours of stimulation with PMA and ionomycin. Numbers in brackets represent total cell numbers c. Statistical analysis of the frequency of IFN+Foxp3+ cells in Tregs stimulated in the presence of vehicle, SF1670, SC-79, 740 Y-P or AS1842856 of n=5 experiments performed. d. Proliferation of CFSE-labeled responder T cells alone (left histogram) or co-cultured with Tregs that have been pre-incubated with either vehicle, SF1670, SC-79 or 740 Y-P at day 4 after co-culture at a 1:2 Treg:Tresp ratio. e. Statistical analysis of the percentage of suppression at different Treg:Tresp ratios of n=6 experiments performed.
AKT isoforms modulate Th1-like Treg generation and function in human autoimmune disease (2016)
Figure 4

Figure 4. Foxo1/3 inhibition is sufficient to induce the Th1 phenotype on Tregs

a. Sorted CD4+CD25hiCD127— were pre-incubated with increasing concentrations of AS1842856, washed and co-cultured with CFSE-labeled responder T cells. Representative histograms of proliferation of Tresp alone (left) or in co-culture with Tregs treated with either vehicle or different concentrations of AS1842856 measured at day 4 at a 1:2 Treg:Tresp ratio. b. Statistical analysis of the suppression of proliferation at different Treg:Tresp ratios (n=6) on Tregs pretreated with vehicle (white dots) or 10 nM (black dots, left diagram) or 50 nM AS1842856 (black dots, right diagram). c. Representative dot plots of the frequency of IFN+Foxp3+ Tregs in the co-cultures in a at a 1:2 Treg:Tresp ratio after adding GolgiStop for the last 6 hours. d. Statistical analysis of the frequency of IFN+Foxp3+ Tregs in the co-cultures in a of n=6 experiments performed. e. Correlation between frequency of IFN+Foxp3+ Tregs in co-cultures and percentage of suppression in Tregs pre-incubated with different doses of AS1842856 at a 1:4 Treg:Tresp ratio. f. IFNG and TBX21 gene expression on Tregs transduced with a Foxo1 or Foxo3 shRNA as compared to the NT-shRNA control, measured at day 6 after 4 hours of stimulation with PMA and ionomycin. g. Percentage of suppression of CFSE-labeled Tresp proliferation co-cultured with Tregs transduced with a non-target (white dots), Foxo1 (left diagram, black dots) or Foxo3 shRNA (right diagram, black dots) at different Treg:Tresp ratios (n=4). *p < 0.05, **p < 0.005, ***p < 0.0005.
AKT isoforms modulate Th1-like Treg generation and function in human autoimmune disease (2016)
Figure 5

Figure 5. Differential roles of AKT isoforms in Th1-Treg generation

Sorted Tregs were stimulated with anti-CD3, anti-CD28, IL-2 and shRNA specific for AKT1, AKT2, AKT3, or non-target control and resorted based on GFP expression at day 5. a. IFNG, TBX21, IL10, FOXP3 gene expression after sort at day 5 (n=5). b. Representative example of staining on resorted Tregs stimulated with PMA and ionomicin for 4 hours at day 5 (n=4). c. Statistical analysis of the frequency of IFN+Foxp3+ Tregs after AKT silencing (n=4). d. shRNA transduced cells were co-cultured with CFSE-labeled Tresp and Tresp proliferation was measured after 4 days. e. Statistical analysis of the percentage of suppression by AKT1-, AKT2- and AKT3-silenced Tregs as compared to NT-transduced cells of n=6 experiments performed. *p < 0.05, **p < 0.005, ***p < 0.0005.
AKT isoforms modulate Th1-like Treg generation and function in human autoimmune disease (2016)
Figure 6

Figure 6. Blockade of the PI3K/AKT/Foxo pathway in Th1-Tregs inhibits IFN secretion and restores their suppressive function

a. Sorted CD4+CD25hiCD127— Tregs were stimulated in the presence of vehicle (white squares) and IL-12 either alone (white circles) or in the presence of LY294002 or MK2206 (black circles) for 3 days and IFNG and TBX21 gene expression was examined every 24 hours. Statistical analysis of n=8 independent experiments performed. b. Representative example of the frequency of IFN+Foxp3+ cells in Tregs stimulated in the presence of vehicle or IL-12 either alone or in the presence of LY294002 or MK2206 for 3 days after a 4 hour stimulation with PMA and ionomycin. c. Statistical analysis of the frequency of IFN+Foxp3+ cells in Tregs stimulated as in c of n=8 experiments performed. d. Proliferation measured at day 4 of CFSE-labeled responder T cells alone (left histogram) or co-cultured with Tregs that had been pre-incubated with either vehicle or IL-12 alone or with LY294002 or MK2206 at a 1:2 Treg:Tresp ratio. e. Statistical analysis of the percentage of suppression as in d at different Treg:Tresp ratios of n=6 experiments performed. *p < 0.05, **p < 0.005, ***p < 0.0005.
AKT isoforms modulate Th1-like Treg generation and function in human autoimmune disease (2016)
Figure 7

Figure 7. Blockade of the PI3K/AKT/Foxo pathway in Tregs from RRMS patients inhibits the Th1-Treg phenotype and restores their suppressive ability

a. mRNA expression on ex vivo isolated Tregs from MS patients and healthy controls (n=15). b. Representative example of the expression of AKT3, Foxo3 and PTEN on ex vivo sorted Tregs from MS patients (black open histogram) and HC (grey open histogram). Grey histograms represent isotype. c. Ex vivo expression of pAKT, pFoxo1 and pFoxo3 in Tregs from RRMS patients (black open histogram) and healthy controls (white open histograms). Grey histograms represent isotype. d. Representative example of the frequency of IFN+Foxp3+ Tregs isolated ex vivo from RRMS patients (lower row) and healthy controls (HC, upper row) and stimulated in the presence of vehicle (left histogram), LY294002 (middle histogram) or MK2206 (right histogram) for 3 days. Staining was performed after a 4-hour stimulation with PMA and ionomycin. e. Statistical analysis of the frequency of IFN+Foxp3+ Tregs in HC (white bars) and RRMS patients (black bars) stimulated in the presence of either vehicle, LY294002 or MK2206 (n=10). f. Representative example of the proliferation of Tresp cells from HC (upper row) and RRMS patients (lower row) either alone (first column) or in co-culture with Tregs that had been previously incubated with either vehicle, LY294002 or MK2206 for one hour and washed to remove drug traces. g. Statistical analysis of the percentage of suppression as in h at different Treg:Tresp ratios of n=8 experiments performed. *p < 0.05, **p < 0.005, ***p < 0.0005.
Constitutive activation of DIA1 (DIAPH1) via C-terminal truncation causes human sensorineural hearing loss (2016)
Figure 2

Figure 2: DIA1(R1204X) induces elongation of microvilli in MDCKmCherry-ESPIN1 cells.

GFP-tagged DIA1, DIA1(M1190D), and DIA1(R1204X) plasmids were transfected into MDCKmCherry-ESPIN1 cells cultured on membrane inserts. Twenty-four hours after transfection, a reconstructed lateral view of fixed cells was generated using a confocal laser fluorescence microscope. Arrows indicate the elongated microvilli visualized by mCherry-ESPIN1 with GFP-hDIA1(R1204X) expression. Scale bars: 5 μm. Representative of 3 experiments.
Constitutive activation of DIA1 (DIAPH1) via C-terminal truncation causes human sensorineural hearing loss (2016)
Figure 3

Figure 3: DIA1(R1204X) induces elongated and thick microvilli in HeLa cells.

Various GFP-tagged DIA1 plasmids were transfected into HeLa cells, and fixed 24 h after transfection. Fixed cells were stained with Alexa568-conjugated phalloidin and DAPI (A, B: representative of 5 experiments).

(A) Stress fiber formation was observed with a confocal laser microscope. Note the induction of stress fibers by GFP-DIA1(M1190D) (asterisks). Arrow and arrowheads indicate a plasma membrane (PM) localization of GFP-DIA1(M1190D) and a dot-like localization of GFP-DIA1(R1204X) adjacent to the PM, respectively. Inset in the top panel of DIA1 (R1204X) shows the magnified image of the region indicated by rectangle. Scale bars: 10 μm..

(B) Microvilli with or without GFP-DIA1(R1204X) expression were observed under a confocal laser microscope. Note elongated and thick microvilli (arrowheads) in GFP-DIA1(R1204X)- transfected cells, but not in non-transfected cells (circles). 3D movies are available in Movie EV1 (R1204X), Movie EV2 (WT), and Movie EV3 (ttaa). Scale bar: 10 μm.
Constitutive activation of DIA1 (DIAPH1) via C-terminal truncation causes human sensorineural hearing loss (2016)
Figure 5

Figure 5: Single-molecule dynamics of the active mutant, hDIA1(R1204X), in XTC cells.

GFP-tagged DIA1(WT), DIA1(R1204X), and DIA1(M1190D) were transfected into XTC cells. XTC cells were spread on the PLL-coated coverslips and time-lapse images were acquired every 300 ms, illuminating the restricted areas near the cell edges, by using a single-molecule speckle microscope. Representative images are shown for DIA1(WT) (A, Movie EV5), DIA1(R1204X) (B, Movie EV6), and DIA1(M1190D) (C, Movie EV7). Circles indicate speckles showing directional movement over a distance of several micrometers. Trajectories of these speckles are also illustrated, and crosses were plotted at the end of the directional movement. Speckles showing directional movement were frequently observed in cells expressing GFP-DIA1(R1204X) (B), whereas such speckles were scarce in cells expressing GFP-DIA1(WT) (A). However, the density of these speckles in GFP-DIA1(R1204X)-expressing cells appeared lower than that in GFP-DIA1(M1190D)-expressing cells, which is considered a fully active mutant (C). To evaluate the frequency of speckles indicating directional movement, we performed a quantitative analysis (D). The number of speckles showing directional movement during 3 continuous planes was counted and were normalized by the fluorescent intensity of each cell, which corresponds to the expression level of the GFP-tagged protein. *P= 0.0288 and **P= 0.0002 Bonferroni’s post hoc test following one-way ANOVA (WT; n = 5, R1204X; n = 6, M1190D; n = 6). Time is in seconds. Scale bars: 5 µm.
Constitutive activation of DIA1 (DIAPH1) via C-terminal truncation causes human sensorineural hearing loss (2016)
Figure 6

Figure 6: No HC loss or hearing loss in Dia1-KO mice.

Cochleae from control, heterozygous Dia1-KO (Dia1-/+) and homozygous Dia1-KO (Dia1-/-) mice were fixed at 5 weeks of age (A, B; representative of n=6).

(A) Paraffin-embedded 4 μm sections of cochlea were stained with hematoxylin and eosin. No hair cell (HC) loss (either IHC or OHC) is observed at the basal turn of the Dia1-/+ or Dia1-/- cochleae. No obvious differences are observed in the spiral ganglion (SV), spiral ligament (SL), stria vascularis (SV), and Reissner’s membrane (RM) in Dia1-/+, or Dia1-/- mice compared with control mice. Lower panels (Scale bars: 20 µm) are magnified images of the region indicated rectangle in upper panels (Scale bars: 100 µm).

(B) Basal turns of the dissected cochleae were immunostained using a Myo7a Ab followed by an Alexa488 secondary Ab with DAPI as a nuclear counterstain. No HC loss (either IHC or OHC) is observed in Dia1-/- mice. Scale bars: 20 µm.

(C) ABR thresholds (10, 20, 40 kHz) in control and Dia1-/- mice at 5 weeks of age (dB SPL, mean SE). No hearing loss was observed in Dia1-/- mice (n = 8) compared with control mice (n = 4).
Constitutive activation of DIA1 (DIAPH1) via C-terminal truncation causes human sensorineural hearing loss (2016)
Figure 7

Figure 7: Expression levels of the transgenic DIA1(R1204X) mutant in the inner ear of TG mice.

(A) Illustration of the linearized expression cassette containing the CAG promoter, 3xFLAG-tagged DIA1(R1204X), and SV40 poly(A) sequence used for injection into fertilized eggs.

(B) Expression of FLAG-tagged DIA1(R1204X) in the inner ear and heart of TG, but not WT, mice is confirmed by immunoblotting using a FLAG Ab. Representative of 3 experiments.

(C) Expression levels of FLAG-tagged DIA1(R1204X) relative to endogenous Dia1 in the inner ear, thymus, and heart were determined by immunoblotting using a Dia1/DIA1 Ab (inner ear; n = 4, thymus and heart; n = 3), and quantified in the inner ear and thymus (*P= 0.0226 and **P= 0.0016 by two-tailed Student’s t-test). Lysates of HEK293 cells overexpressing equal amounts of GFP-Dia1 and GFP-DIA1 (determined using a GFP Ab: right end) are detected with equal affinity by the Dia1/DIA1 antibody. Comparable loading of proteins is confirmed using an α−tubulin or GAPDH Ab.
Constitutive activation of DIA1 (DIAPH1) via C-terminal truncation causes human sensorineural hearing loss (2016)
Figure 8

Figure 8: DIA1(R1204X)-TG mice show progressive hearing loss and a reduction of HC.

(A), Age-related click and pure tone-burst (8, 16, 24, 32 kHz) ABR thresholds (dB SPL, mean SE) in DIA1(R1204X)-TG (5 weeks, n = 6; 10 weeks, n = 16; 25 weeks, n = 42) and control mice (5 weeks, n = 6; 10 weeks, n = 16; 25 weeks, n = 33). Note the progressive hearing loss of DIA1(R1204X)-TG mice starting at high frequency sounds from 10 weeks. **P= 0.0039 (24 kHz) and **P< 0.0001 (32 kHz) at 10 weeks and **P< 0.0001 (click), **P< 0.0001 (8 kHz), **P= 0.0001 (16 kHz), **P< 0.0001 (24 kHz), and **P< 0.0001 (32 kHz) at 25 weeks by Bonferroni’s post hoc test following two-way ANOVA.

(B) Representative SEM images of the organ of Corti at the basal turn obtained from DIA1(R1204X)-TG and control mice at 5, 10, and 25 weeks of age. White and red circles show IHC and OHC losses, respectively. Note OHC-dominant HC loss. Scale bars: 5 µm.

(C) The percentages of remaining OHC and IHC in each turn in DIA1(R1204X)-TG and control mice at 25 weeks of age (n = 5). Schematic illustration shows the regions of the cochlea divided into three parts: Ap; apical turn, Md; middle turn, Bs; basal turn. **P= 0.0017 (OHC at Md), **P< 0.0001 (OHC at Bs), *P= 0.0117 (IHC at Ap), *P= 0.0330 (IHC at Md), and **P= 0.0003 (IHC at Bs) by Bonferroni’s post hoc test following two-way ANOVA.

(D) DPOAE (f2 frequency at 8, 12, 16, 20 kHz) amplitude (dB SPL, mean SE) in DIA1(R1204X)-TG (n = 37) and control mice (n = 30) at 25 weeks of age. *P= 0.0127, **P= 0.0029 (at 12 kHz), and **P= 0.0013 (at 16 kHz) by Bonferroni’s post hoc test following two-way ANOVA.

(E) High magnification SEM images of OHCs and IHCs at the basal turn of cochlea obtained from DIA1(R1204X)-TG and control mice at 25 weeks of age. Note the sparse and short (arrow) stereocilia of OHC, and sparse and fused (arrowheads) stereocilia of IHC in DIA1(R1204X)-TG mice. Scale bars: 1 µm in OHCs and 5 µm in IHCs.
Aut2p and Aut7p, two novel microtubule‐associated proteins are essential for delivery of autophagic vesicles to the vacuole (1998)
Figure 2

(A) After 4 h of starvation in 1% K‐acetate medium in the presence of PMSF, autophagic vesicles can be visualized in vacuoles of wild‐type and pep4Δ prb1Δ cells using Normaski optics. In aut2Δ and aut7Δ cells no autophagic vesicles can be seen. Bar represents 20 μm. (B) Total protein breakdown of cells starved on nitrogen‐free medium is significantly reduced in aut2Δ and aut7Δ cells compared with wild‐type cells. pep4Δ (pra1Δ) cells are almost completely impaired in starvation‐induced vacuolar protein breakdown. Cells were pulse‐labeled with [35S]methionine and chased on non‐radioactive starvation medium. Aliquots were taken at the indicated times and acid soluble small peptides generated by proteolysis were determined according to Straub et al. (1997). (C) Survival during starvation. Like pep4Δ (pra1Δ) cells, which are almost completely impaired in vacuolar proteolysis, aut2Δ and aut7Δ cells exhibit a significantly reduced survival rate during starvation on nitrogen‐free medium as compared with wild‐type cells. Aliquots of cells incubated in 1% K‐acetate were plated and growing colonies were determined according to Straub et al. (1997).
Aut2p and Aut7p, two novel microtubule‐associated proteins are essential for delivery of autophagic vesicles to the vacuole (1998)
Figure 3

Maturation of proaminopeptidase I is impaired in aut2‐1 (A), aut2Δ (B) and aut7Δ (C) cells. Further details are outlined in the text. Crude extracts were prepared from cells starved 4 h on 1% K‐acetate.
Aut2p and Aut7p, two novel microtubule‐associated proteins are essential for delivery of autophagic vesicles to the vacuole (1998)
Figure 5

(A) Detection of protein–protein interactions using the two‐hybrid system. Intense staining indicates an interaction between Aut2p and Aut7p, Aut2p and Tub1p, and Aut2p and Tub2p. AUT7, TUB1 and TUB2 are cloned in‐frame with the GAL4‐activator domain. AUT2 and AUT7 were fused to the GAL4‐binding domain (see Materials and methods). (B) Affinity chromatography confirms protein–protein interaction between Aut2p and Aut7p. A fusion protein of Aut2p with the 26 kDa glutathione‐S‐transferase (GST) domain from Schistosoma japonicum (see Materials and methods) was bound to glutathione–Sepharose 4B. This GST–Aut2p column was incubated with a crude extract from aut7Δ cen Met25::AUT7–GFP cells. The non‐bound supernatant (S) fraction (lane 1) was taken away. Gel beads were successively washed with NaCl solutions of increasing concentrations to remove non‐specifically bound proteins. After the final 500 mM NaCl wash (W, lane 2), bound proteins were eluted with Laemmli buffer (E, lane 3). Samples were analyzed in immunoblots with monoclonal antibodies directed against GFP. Using Aut2p–GST, a clear band corresponding to Aut7p–GFP is seen in the eluate fraction (lane 3, arrowhead). As a control, GST alone was attached to the column and no band was detectable (lane 6). (C) As described in (B), bovine tubulin binds to GST–Aut2p glutathione–Sepharose 4B (lane 12), but not to GST glutathione–Sepharose 4B (lane 9). Protein staining with Coomassie is shown in lanes 1–6; in lanes 7–12 tubulin was detected using monoclonal antibodies (Boehringer Mannheim). Further details are given in Materials and methods. (D) Similar to (C), bovine tubulin does not bind to GST–Aut7p glutathione–Sepharose 4B (lane 12) and to GST glutathione–Sepharose 4B (lane 9). Lanes 1–6, protein staining with Coomassie; lanes 7–12 probed with antibodies directed against tubulin.
Aut2p and Aut7p, two novel microtubule‐associated proteins are essential for delivery of autophagic vesicles to the vacuole (1998)
Figure 6

Benomyl sensitivity of wild‐type cells overexpressing AUT2 and AUT7, and of aut2Δ and aut7Δ cells is not altered. Cells were grown to stationary phase and dilution series with decreasing cell densites were dropped on CM plates without uracil containing 15 and 30 μg/ml benomyl. Plates were incubated at 30°C.