EMBO/sd-character-level-ner huggingface dataset:
Endothelial specific PAI-1 inactivation prevents age-associated glomerular lesions.Figure 5A: PAI-1 immunohistochemistry (upper panels) and quantification of PAI-1-positive glomeruli (lower panel) in kidneys of PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 4 for PAI-1flox and PAI-1∆endo mice, respectively. B: Morphology (PAS staining, upper panels) and quantification of glomerular lesions (lower panel) of kidneys from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 12 for PAI-1flox and PAI-1∆endo mice, respectively. C: TUNEL assay (upper panels) and quantification of TUNEL-positive glomeruli (lower panel) in kidneys from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 6 for PAI-1flox and PAI-1∆endo mice, respectively. D: WT1 immunostaining (upper panels) and quantification of WT1-positive glomerular cells (lower panel) in kidneys from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 4 and n = 6 for PAI-1flox and PAI-1∆endo mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: PAI-1∆endo versus PAI-1flox mice. E: Serum creatinine measurement in PAI-1flox (n = 6) and PAI-1∆endo (n = 7) mice at 22 months of age. Data information: Data are means ± SEM. Statistical analysis: t-student test: PAI-1∆endo versus PAI-1flox mice. F: Senescence-associated β-galactosidase staining (upper panels) and quantification of β-galactosidase-positive glomeruli (lower panel) in kidneys from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 4 for PAI-1flox and PAI-1∆endo mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: PAI-1∆endo versus PAI-1flox mice. G: p21/griffonia simplicifolia/nephrin coimmunostaining in kidneys (upper panels) from PAI-1flox and PAI-1∆endo mice at 22 months of age and quantification (lower panel) of p21-positive endothelial cells per glomeruli. Original magnification X630. Scale bar = 10 μm. n = 4 and n = 6 for PAI-1flox and PAI-1∆endo mice, respectively. H: 53BP1/CD34 coimmunostaining (upper panels) and quantification of glomerular 53BP1 foci in glomerular endothelial cells from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X630. Scale bar = 10 μm. n=4 and n=6 for PAI-1flox and PAI-1∆endo mice, respectively. Arrows show 53BP1 positive foci. Data information: Data are means ± SEM. Statistical analysis: t-student test: PAI-1∆endo versus PAI-1flox mice.
Figure 1(A) Schematic of PP7-tagged RNA reporters and PCP-NLS-FLAG constructs used in this study. (B) Relative protein expression from PP7-tagged CGG-nLuc reporters compared to AUG-driven reporters in HEK293T cells (n=12 biological replicates). Data information: error bar represents mean +/- SD. Statistical analysis was performed using two-tailed Student's t test with Welch's correction, ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. (C) Expression of AUG-control and CGG-nLuc RAN translation reporters in HEK293T cells treated with the ER stress agent thapsigargin (TG, 2 μM) (n=11 biological replicates) normalized to vehicle (DMSO). Data information: error bar represents mean +/- SD. Statistical analysis was performed using two-tailed Student's t test with Welch's correction, ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. (D) Schematic of immunoprecipitation and mass spectrometry experiments aimed at identifying CGG repeat RNA interacting proteins Growth media, containing different isotopes of lysine and arginine for SILAC labeling, are indicated as light (Lys-0 and Arg-0) and medium (Lys-4 and Arg-6). Log2 fold-change of the CGG-interacting protein enrichment compared AUG-reporter (n=2 independent experiments; error bars represent range between repeats) under normal (E) Log2 fold-change of the CGG-interacting protein enrichment compared AUG-reporter (n=2 independent experiments; error bars represent range between repeats) under normal and after integrated stress response activation by TG (F). (G) List of top proteins enriched in CGG-PP7 RNA interaction compared to AUG in mass spectrometry experiments without or with TG treatment (TG+). Bar graphs represent average of two biological replicates +/- range. Enriched SR proteins are marked with blue dots.
a, b, Oral glucose tolerance test (OGTT) before (a, week 0) and after (b, week 4) 4 weeks of HFD.f4a, b, Oral glucose tolerance test (OGTT) before (a, week 0) and after (b, week 4) 4 weeks of HFD. c, d, OGTT (c) and serumleptin and adiponectin levels (d) after 8 weeks of daily exercise. For a-d, results represent the mean ± s.e.m. for 4-5 mice per group. E, exercise; NE, no exercise; RD, regular diet . *P  0.05, **P  0.01, (c, one-way ANOVA; d, Wilcoxon rank test). NS, not significant.
cfDNA-positive PDAC present increased lethality and metastatic dissemination.Figure 5A, Representative images of PI3K activation (as shown by positive pAkt substrate staining) on the primary tumours of PDAC patients, with values of quantification of cfDNA and mutated KRAS allele frequency. B, Quantification of pAkt substrate staining in T3 patients (total number of patients = 6, n = 2 in each group). Mean +/- SEM. C, Survival curve of KPC mice presenting different levels of cfDNA. n in each group is indicated. Log rank test, *** p<0.001 D, Quantification of cfDNA in KPC mice at different stages of the disease. n in each group is indicated. PanIn= Pancreatic Intraepithelial Neoplasia. Non parametric Mann Whitney, * p<0.05, ** p<0.01, *** p<0.001.
Figure 4(A) Table showing examples of the selectivity of all plastid GNATs on a selection of retrieved N-termini. exhaustive. The ten first amino acids of the N-α-acetylated proteins are indicated. The color code is green, positive; red, negative; grey means that the data is missing i.e. that the peptide was not quantified. (B) IceLogo representation of the N-termini substrates of GNAT2 vs all that of other GNATs. To construct the dataset, all GNAT2 substrates with NTA yield threshold >30 % were selected in the positive set. The negative set corresponded to the compilation of all substrates with a threshold >30 % of all other seven GNATs. The color symbol is associated to the default choice, which the software proposes for each class of amino acid: Green is for the class of small hydrophilic uncharged residues including S, T, G. Acidic residues including D or E are colored in red. Positively- charged residues including K, R and H are displayed in blue. N or Q are in purple. Hydrophobic residues (A, I, M, V, L, W, Y, F, P) are shown in black.
Figure.Figure 7A. Schematic illustration of the experimental procedures.  We combined the live-cell imaging, blastomere biopsy and single-cell RNA sequencing to accurate analysis. B. Heatmap comparing ZGA genes expression between WT-2 and NT-2 and si6B-NT embryos (FC > 5, FPKM > 5 in each replicate; left). A total of 1,813 DEGs are classified into two groups by unsupervised hierarchical clustering. KEGG and GO analysis of the two groups by unsupervised hierarchical clustering (right). C. Scatter plots comparing the si6B-NT and NT-2 genes expression. The higher expression genes in siRNA-6B and NT-2 are colored with red and blue, respectively (FC > 5, FPKM >5). D. MA plot comparing gene expression between si6B-NT and NT-2. The data analyzed derive from two independent biological replicates. Arrows represent data points outside of the plotting area. DEG, differentially expressed genes. E. Venn diagram showing the overlap between the genes that failed to be activated in SCNT 2-cell embryos and derepressed in knock-down KDM6B (left). Heat-map, KEGG and GO enrichment showing the expression pattern of 319 overlap genes (FC > 5, FPKM >5; right). F. Schematic illustration of the experimental approach. G. Representative localization of Xist expression in the nuclei of SCNT embryos injected with siRNA-6A, siRNA-6B or siRNA-Xist (left). Arrows indicate the blastomeres enlarged in the bottom panels. The ratios of blastomeres classified according to the positive or negative expression of Xist analyzed by RNA-FISH (right). Each bar represents a single embryo. H. Large-scale qPCR analysis of Xist and eight X-linked genes in 4-cell stage male embryo. Each rectangle bar represents the expression value detected by single embryo RT-qPCR. The number of embryos in each group is indicated. Coloured bars indicate expression levels. I. Image of full-term cloned pups derived from NT Sertoli cells injected with siRNA-control, siRNA-Xist, or siRNA-Xist-6B.
Figure 2A. Representative images of mouse hippocampal neurons transfected with either GFP-expressing control or Lrig1 shRNA vector at 9 DIV and maintained for 3 additional days in vitro (9+3 DIV). Scale bar, 15 m. Boxed area represents a higher-magnification image showing the profuse proximal dendritic arborization of Lrig1-shRNA transfected neurons.B. Sholl analysis of the dendritic arbor from hippocampal neurons transfected with either control or Lrig1 shRNA-GFP vector at 9 DIV and maintained for 3 additional days in vitro (9+3 DIV). Data are shown as mean SEM of n=3 independent experiments. *p<0.05 and **p<0.01 by two-way ANOVA followed by Bonferroni multiple comparisons test.C-G. Quantification of primary (C), and secondary (D) dendrites as well as total dendritic branching (E), terminal dendritic points (F), and total dendritic length (G) of hippocampal neurons transfected with either control or Lrig1 shRNA-GFP vector. The results are shown as mean SEM of n=3 independent experiments. *p<0.05 by Students t test. H. Knockdown efficiency was analyzed by real-time RT-PCR in MN1 cells transfected with control or Lrig1 shRNA vectors. Transfected cells were enriched by puromycin treatment in order to increase the population of cells expressing control or Lrig1 shRNA constructs. Data are shown as individual values of a representative assay measured in triplicates. n=2 independent experiments were performed. I. Representative images of MAP-2 immunostained hippocampalneurons obtained from wild-type and Lrig1-deficient mice cultured for 7 days in vitro (7 DIV). Scale bar, 15 m.J. Sholl analysis of the dendritic arbor from MAP-2 stained hippocampalneurons (7 DIV) isolated from wild type and Lrig1-deficient mice. Data are shown as mean SEM of n=3 independent experiments. *p<0.05 by two-way ANOVA followed by Bonferroni multiple comparisons test.K-M. Quantification of the number of primary dendrites (K), secondary dendrites (L) and total dendritic branching (M) from MAP-2 stained hippocampalneurons (7 DIV) isolated from wild-type and Lrig1-deficient mice. The results are shown as mean SEM of n=3 independent experiments. *p<0.05 by Students t test.
Figure 5The structures of SARS-CoV-2 RBD, GD/1/2019 RBD and GX/P2V/2017 RBD displayed in surface view. Residues that interact with the hACE2 are marked. Sequence alignment of the RBD sequences from the SARS-CoV-2, GD/1/2019 and GX/P2V/2017. Red triangles indicate the two residue substitutions at the binding sites of both GD/1/2019 RBD and GX/P2V/2017 RBD. Blue triangles indicate the five other residue substitutions at the binding site of GX/P2V/2017 RBD. Identical residues are highlighted in red background, and similar residues are labelled in red and boxed in blue lines. The green Arabic numerals 1-3 indicate cysteine residues that pair to form disulfide bonds.
Figure 5qRT-PCR detection of IFNB1 mRNA in WT and CGAS KO THP-1 cells stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly (I:C) for 4 h. ELISA detection of IFNβ in supernatants of WT and CGAS KO THP-1 cells stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly(I:C) for 4 h. qRT-PCR detection of Ifnb1 mRNA in mBMDMs from WT and Cgas-/- (Cgas KO) mice stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly(I:C) for 4 h. ELISA detection of Ifnb1 in supernatants of mBMDMs from WT and Cgas KO mice stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly(I:C) for 4 h. qRT-PCR detection of IFNB1 mRNA in WT and CGAS KO THP-1 cells stimulated with ecGAMP (5 μg/ml) or icGMAP (0.1 μg/ml) or transfected with ISD. qRT-PCR detection of Ifnb1 mRNA in mBMDMs from WT and Cgas KO stimulated with ecGAMP (5 μg/ml) or icGMAP (0.1 μg/ml) or transfected with ISD. Western blot detection of indicated proteins in lysates of mBMDMs from WT and Cgas KO mice stimulated with ecGAMP (5 μg/ml), icGAMP (0.1 μg/ml) or transfected with ISD. Data are representative of 3 independent experiments. qRT-PCR detection of IFNB1 mRNA of WT, CGAS KO, CGAS KO complemented with cGAS (CGAS KO+cGAS) or CGAS KO stably transfected with the empty vector (CGAS KO+Vector) THP-1 cells stimulated with ecGAMP (5 μg/ml). ELISA detection of IFNβ in supernatants (I) of WT, CGAS KO, CGAS KO complemented with cGAS (CGAS KO+cGAS) or CGAS KO stably transfected with the empty vector (CGAS KO+Vector) THP-1 cells stimulated with ecGAMP (5 μg/ml).
Figure 6A. Nutrient starvation reduced the level of LRP6 in mouse liver. Mice were starved for 36 h and liver tissues were analyzed by immunoblotting (left panel). The ratio of LRP6/Vinculin of three independent immunoblots was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. **P < 0.01. Student's t-test was used for statistical analysis. B. Refeeding of nutrients rescued LRP6 in mouse liver. Mice were starved and then refed for 6 h and liver tissues were analyzed by immunoblotting (left panel). The ratio of LRP6/Vinculin of three independent immunoblots was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. *P < 0.05. Student's t-test was used for statistical analysis. C. The level of LRP6 was not reduced during nutrient starvation in mouse brain. Mice were starved and brain tissues were analyzed by immunoblotting. D. LRP6 was O-GlcNAcylated in mouse liver. Liver tissues were incubated with sWGA-beads in the absence or presence of free GlcNAc (25 mM) for 3 h. After incubation, sWGA-beads were precipitated and analyzed by immunoblotting. WCL, whole-cell lysates. E. O-GlcNAcylation of LRP6 was reduced during starvation in mouse liver. LRP6 in liver tissue lysates from both fed and starved mice was equilibrated by loading twice as much lysate from the livers of starved mice on the gel. Liver tissues were incubated in sWGA-beads for 3 h. After incubation, sWGA-beads were precipitated and analyzed by immunoblotting. WCL, whole-cell lysates.
Figure 6A. BMDM cells were infected with VSV (MOI, 0.1) or HSV (MOI, 5) for the indicated times. Cell lysates were analyzed by immunoblotting with the indicated antibodies. B. BMDM cells were transfected with control or TBK1 siRNA. 72 hours after transfection, cells were analyzed by immunoblotting with antibody for phospho-RKIP or TBK1. C. Immunoblot analysis of RKIP phosphorylation in control or TBK1-/- mouse embryonic fibroblasts after VSV (MOI, 0.1) infection for the indicated times. D. Recombinant flag-tagged TBK1 was immunoprecipitated with anti-flag M2 beads in flag-TBK1 transfected 293T cells. Followed by in vitro kinase assay, which was carried out in the reaction mixture containing 2 mM ATP, 100 μM GST-RKIP or 100 μM GST-GFP together with 5 μM flag-TBK1 at 30 °C for 30 min. Assay mixtures were immunoblotted with the indicated antibody for phospho-RKIP, flag, GST or his. E. Immunoblot analysis of extracts of RAW264.7 cells stably overexpressing flag-RKIP, flag-S109A and flag-S109D, infected with VSV (MOI, 0.1) for the indicated times, followed by immunoprecipitation with anti-flag beads. F. BMDM cells were infected with VSV (MOI, 0.1) for the indicated times. The cell lysates were immunoprecipitated with anti-TBK1 (left) or anti-RKIP (right). The immunoprecipitates were analyzed by immunoblot with anti-RKIP or anti-TBK1 antibody. The levels of the endogenous RKIP, TBK1, and p-RKIP were detected by immunoblot analysis. G. Co-immunoprecipitation and immunoblot analysis of 293T cells transfected with myc-TBK1 alone or with flag-RKIP, flag-S109A or flag-S109D. H. GST-pull down experiment was performed with 1 μg fusion protein GST-GFP, GST-his-RKIP or GST-his-S109D mixed with 20 μl precleared agarose beads in 800 μl reaction medium, followed by adding 1 μg flag-TBK1 and incubating at 4°C for 3 hours with gentle rotation. Pull down (1st-3th lane) and input samples (4th lane) were separated by SDS-PAGE followed by immunoblotting with anti-GST or anti-flag antibody. I. Immunoprecipitation and immunoblot analysis of 293T cells transfected with indicated combinations of vector for myc-TBK1, flag-RKIP, flag-S109A, flag-S109D and HA-GSK3β or flag-GFP. J. In vitro kinase assay was carried out in the reaction mixture containing 2 mM ATP, 50 μM GST-TBK1 together with 100 μM GST-his-RKIP, 100 μM GST-his-S109D, 100 μM GST-his-S109A or 100 μM GST, respectively. React at 30 °C for 30 min. Then followed by immunoblotting with anti-p-TBK1or anti-GST antibody.
in vivo recapitulation of the molecular defects caused by elevated DHS levels lead to severe decline of cardiac functionality which can be prevented by HAT inhibition: (A) Schematic representation of the experimental setup employed in the in vivo mouse experiments.Figure 6(A) Schematic representation of the experimental setup employed in the in vivo mouse experiments. Representative micrographs depicting (B) γH2A.X staining on the ventricle sections for the indicated cohorts. Representative micrographs depicting (C) H3K27ac staining on the ventricle sections for the indicated cohorts. (D) Quantification of γH2A.X+ CMs nuclei represented as percentage bar graph. (E) Box-plot depicting the distributions of the corrected total nuclear fluorescence of the indicated cohorts. (F) Representative micrographs depicting ventricular view from the echocardiography of the indicated cohorts. (G-H) Quantification of fractional shortening and ejection fraction of the indicated groups represented as bean plot and percentage bar graph respectively (n=6 animals per condition). Scale bars for B,C = 50 µm.
Fig.Figure 4A) Native mass spectrum of NHE9* purified in the presence (top panel) or the absence of additional brain lipids (bottom panel). In the presence of brain lipids, NHE9* is detected as exclusively dimeric protein with poorly resolved peaks, indicating the presence of multiple lipid adducts. Without brain lipid addition, NHE9* appears as sharp peaks, revealing several adducts of approximately 1 kDa each, as well as a minor monomer population. B) Thermal stabilization of purified dimeric NHE9*-GFP by lipids. Normalized mean FSEC peak fluorescence before heating (open bars), and after heating and centrifugation (grey bars) in presence of different lipids. Data presented are mean values ± data range of n = 4 experiments (see Materials and Methods). C) Thermal shift stabilization of purified dimeric NHE9*-GFP in the presence of PIP2 (red) compared to PIP2-free (black). Data presented are normalized mean FSEC peak fluorescence as mean values ± data range of n = 2 technical repeats; the apparent Tm was calculated with a sigmoidal 4-parameter logistic regression function; the average ΔTm presented is calculated from n = 2 independent titrations. D) Cartoon representation of NHE9 including a model of the loop between TM2-TM3, coloured as in Fig. 2A. The positively charged residues Lys85, Lys105 and Lys107 are shown as purple spheres. E) Thermal shift stabilization of the dimeric NHE9*-K85Q-K105Q-K107Q-GFP by PIP2 (red) compared with thermal shift in the absence (black) of PIP2; data shown are mean values ± data range of n = 2 technical repeats; the apparent Tm was calculated with a sigmoidal 4-parameter logistic regression function; the average ΔTm presented is calculated from n = 2 independent titrations.
Loss of Sinhcaf leads to an increase in histone 3 acetylation of FG follicles.Figure 4(A, B and C) Co-immunoprecipitation of zebrafish Sinhcaf-GFP with HA-Hdac1 (A), HA-Sin3a (B) or HA-Sap18 (C) in HEK293Tcells transfected with both Sinhcaf-GFP and HA-Hdac1 (A), HA-Sin3a (B) or HA-Sap18 (C) plasmids. (D) Relative mRNA level of sinhcaf in zebrafish PG, PV, EV, MV and FG follicles was measured using RT-qPCR. Data shown are mean ± SEM (n = 3, biological replicates). Data information: EV, early vitellogenic stage (early stage III); FG, full-grown stage (late stage III); MV, mid vitellogenic stage (mid stage III); PG, primary growth (stage I); PV, previtellogenic stage (stage II). (E and F) Western blots for acetyl-histone H3 (K9) (Ace-H3-K9), histone H3 in WT (E) or MZsinhcaf -/- (F) PG, PV, EV, MV and FG follicles. α-tubulin was used as a loading control. Data information: EV, early vitellogenic stage (early stage III); FG, full-grown stage (late stage III); MV, mid vitellogenic stage (mid stage III); PG, primary growth (stage I); PV, previtellogenic stage (stage II). (G and H) Statistical analysis of the relative levels of Ace-H3-K9 in WT (G) or MZsinhcaf -/- (H) PG, PV, EV, MV and FG follicles. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. The different letters (a, b, c) indicate a significant difference between the means, p<0.05. Data information: EV, early vitellogenic stage (early stage III); FG, full-grown stage (late stage III); MV, mid vitellogenic stage (mid stage III); PG, primary growth (stage I); PV, previtellogenic stage (stage II). (I, J and K) Western blots for Ace-H3-K9 and H3 in WT and MZsinhcaf -/- EV (I), MV (J) and FG (K) follicles. α-tubulin was used as a loading control. Data information: EV, early vitellogenic stage (early stage III); FG, full-grown stage (late stage III); MV, mid vitellogenic stage (mid stage III) (L) Western blots for Ace-H4-K5, Ace-H3-K18 and H3 in WT and MZsinhcaf -/- FG follicles. α-tubulin was used as a loading control. Data information: FG, full-grown stage (late stage III) (M, N and O) Statistical analysis of the relative levels of Ace-H3-K9I n WT and MZsinhcaf -/- EV (M), MV (N) and FG (O) follicles. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using unpaired two-tailed Student's t-test. Data information: EV, early vitellogenic stage (early stage III); FG, full-grown stage (late stage III); MV, mid vitellogenic stage (mid stage III) (P and Q) Statistical analysis of the relative levels of Ace-H3-K18 (P) and Ace-H4-K5 (Q) in WT and MZsinhcaf -/- FG follicles. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using unpaired two-tailed Student's t-test. Data information: ; FG, full-grown stage (late stage III)
a, Silver staining of LC3 complexes purified from U2OS cells expressing ZZ–Flag–LC3 in normal medium or Earle’s balanced salt solution (EBSS) for 2 h.f1a, Silver staining of LC3 complexes purified from U2OS cells expressing ZZ-Flag-LC3 in normal medium or Earle's balanced salt solution (EBSS) for 2 h. Asterisks mark centriolar satellite proteins. b-d, Co-immunoprecipitation (IP) of OFD1 with LC3, LC3 with PCM1, or OFD1 with PCM1 in HEK293T cells. HA, haemagglutinin. e, Western blotting analysis of PCM1 and OFD1 protein levels in control or PCM1 knockdown (KD) HEK293T cells. f, Co-immunoprecipitation of OFD1 with LC3 in control or PCM1 KD HEK293T cells. IP efficiency is calculated as the ratio of immunoprecipitated OFD1/LC3. g, h, Western blotting analysis of protein levels of indicated proteins in MEFs with indicated genotypes in normal medium or subjected to 24 hserum starvation (SS). Quantified OFD1 level was normalized with alpha-tubulin. Similar results were observed in three independent experiments. g, h, Western blotting analysis of protein levels of indicated proteins in MEFs with indicated genotypes in normal medium or subjected to 24 h serum starvation (SS), 50 nM bafilomycin A1 (Baf), 20 µM chloroquine (CQ) or 1 µM MG132. Quantified OFD1 level was normalized with alpha-tubulin. Similar results were observed in three independent experiments.