EMBO/sd-character-level-ner huggingface dataset:
The apical loop of the HCV IRES domain II change the position at the E-site during initiation.Figure 7(A) Comparison of HCV IRES from 80S*HCV IRES Classical (pink) and tRNAs from POST state (gray)(PDB 5AJ0) (Behrmann et al, 2015) in a common 40S body alignment of the cryo-EM maps.(B) Same as above but in a common 40S head alignment. (C) Comparison of the 40S subunit and bound ligands of the classical 80S*HCVIRES complex (orange) and 80S*HCVIRES*Met-tRNA*eIF5B*GMPPNP complex (POST-like state) (yellow)(EMD 2683) (Yamamoto et al, 2014) in common 60S alignment.(D) The models of the 80S*HCVIRES Classical complex (gray), 80S*HCVIRES*Met-tRNA*eIF5B*GMPPNP complex (POST-like state) (green)(PDB 4UPZ) (Yamamoto et al, 2014) and 80S*HCVIRES Classical/no head tilt complex (pink) are rigid body fitted into the corresponding densities in a common 40S body alignment. The Helix 68 from the 28S rRNA is depicted as well (blue).
Figure 7(A) 786-0 cells lacking GDH1 with restored expression of WT, ED or 3KR mutant GDH1 were subjected to aa starvation for 24 hours. PI/Annexin V double staining of the indicated cells was performed, and the number of apoptotic cells in each group was calculated. The data are represented as the mean ± SEM of three independent experiments. Unpaired t-test was used with **p < 0.01. Xenogeneic inoculation of 786-0 cells lacking GDH1 with restored expression of WT, ED or 3KR mutant GDH1 into the left groin of nude mice. The solid tumor formed by the indicated 786-0 cells is shown (B). Xenogeneic inoculation of 786-0 cells lacking GDH1 with restored expression of WT, ED or 3KR mutant GDH1 into the left groin of nude mice. The tumor growth rate were recorded and analyzed. Data information: All statistical analyses were conducted using unpaired t-test (n = 6). (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001). Xenogeneic inoculation of 786-0 cells lacking GDH1 with restored expression of WT, ED or 3KR mutant GDH1 into the left groin of nude mice. , tumor weight (D) and αKG levels in tumors (E) were recorded and analyzed. Data information: All statistical analyses were conducted using unpaired t-test (n = 6). (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001). Xenogeneic inoculation of 786-0 cells lacking GDH1 with restored expression of WT, ED or 3KR mutant GDH1 into the left groin of nude mice. The tumors were collected to extract total RNA, and qPCR was used to test the mRNA level of the indicated RPL/S genes (F). Xenogeneic inoculation of 786-0 cells lacking GDH1 with restored expression of WT, ED or 3KR mutant GDH1 into the left groin of nude mice. The staining of Ki67 (G, upper panel) and TUNEL (G, lower panel) using tumors from (B) are shown and analyzed (G, right panel). We used 6 mice in each group. Scale bars: 50 μm. Data information: All statistical analyses were conducted using unpaired t-test (n = 6). (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001). (H) Schematic model of the αKG-mediated regulation of RP genes. We uncovered the mechanism by which KIRC cells respond to aa starvation and regulate their protein synthesis to save limited nutrients. Specifically, in KIRC cells under aa starvation, GDH1 is transferred from the mitochondria to the cytoplasm and degraded by the ubiquitin degradation pathway mediated by the E3 ligase RNF213. The degradation of GDH1 directly leads to decreased intracellular αKG, causing the weakened activity of KDM. H3K9me3 and H3K27me3 were demethylated by KDM4 and KDM6, particularly KDM4A and KDM6A, which occupied the promoter of RP genes and cooperatively downregulated the expression of the RP gene during aa starvation. The tumor cells can then preserve the remaining amino acids for survival. Notably, because RP genes are regulated by operons in prokaryotes, we revealed that the expression of most RP genes in renal cell carcinoma cells is mediated by the metabolite αKG.
Figure 2(A) Acyl-RAC assay of HEK293T cells transfected with Flag-cGAS 24 h after transfection with the indicated shRNA. (B) Quantification of palmitoylation levels of Flag-cGAS in (A). Data information: (C) Acyl-RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h. HAM: hydroxylamine. (D) HEK293T cells were transfected with HA-cGAS (WT or C474S mutant) (200 ng), Flag-STING (200 ng) and Myc-ZDHHC18 (200 ng) expression plasmids for 24 h before luciferase reporter assays. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; **, P˂0.005; ***, P˂0.001 (D (E and F) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with the indicated plasmids for 24 h. Data information: WCL: whole-cell lysate; IP: immunoprecipitation; FL: full length (G) Schematic diagrams of ZDHHC18 and its truncation mutants. Data information: TM: transmembrane domain. (H) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with plasmids encoding HA-cGAS and Flag-ZDHHC18 truncations or GFP for 24 h. Data information: WCL: whole-cell lysate; IP: immunoprecipitation; FL: full length (I) Schematic diagrams of cGAS and its truncation mutants. (J) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with plasmids encoding HA-ZDHHC18 and Flag-cGAS truncates or GFP for 24 h. Data information: WCL: whole-cell lysate (K) Immunofluorescence analysis of HA-cGAS (green) and Flag-ZDHHC18 (red) in HT-DNA-stimulated (or not) HeLa cells. Scale bars: 7 μm. (L) Colocalization (merged volume of total cGAS signal) of cGAS and ZDHHC18 in (K). Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; **, P˂0.005; ***, P˂0.001 L). (M-N) Comparison of the binding free energy (M) and binding energy decomposition of a per-residue to the binding affinity between ZDHHC18 and cGAS (N) in different complexes.
Figure 6(A) Schematic showing the method used to generate FOXM1+/+ and FOXM1-/- human NPCs. (B) Representative cases of FOXM1+/+ and FOXM1-/- NPCs clones. Scale bar, 20μm. (C) Graph shows the number of cells per clone in FOXM1+/+ and FOXM1-/- human NPCs (FOXM1+/+ n=12; FOXM1-/- n=13; at least three biological replicates. two-tailed unpaired t-test, P= 0.0014). ( (D) Overexpression of FOXM1 or LIN28A rescues the human neural progenitor cell reduction in FOXM1-/- cell lines. Western blot analysis for SOX2, PAX6 and HOPX. β-actin was detected as loading control. (E) Statistics of relative intensity of SOX2, PAX6 and HOPX (PAX6: n=3 biological replicates; one-way ANOVA, F (2,6) = 8.716, FOXM1-/-(Control) vs. FOXM1-/-(hFOXM1), P= 0.0173, FOXM1+/+(Control) vs. FOXM1-/-(hFOXM1), P= 0.0476. SOX2: n=5 biological replicates; one-way ANOVA, F (2, 12) = 14.72, FOXM1+/+(Control) vs. FOXM1-/-(Control), P= 0.0015, FOXM1+/+(Control) vs. FOXM1-/-(hFOXM1), P=0.9906. HOPX: n=4 biological replicates, one-way ANOVA F (2, 9) = 25.28, FOXM1+/+(Control) vs. FOXM1-/- (Control), P= 0.0018, FOXM1+/+(Control) vs. FOXM1-/-(hFOXM1), P= 0.2211). ( (F) The model for the role of hFOXM1 in cerebral cortex. Human FOXM1 expression causes neural precursor cells increase through up-regulating the level of LIN28A. The increased neural precursor cells subsequently increase in the number of new born neurons and induce cortical extension and folding in mice.
Figure 8Mice were injected with control (Ctrl) or STING-KO MC38 cells, and subjected to treatments, with each dose consisting of 25 mg/kg, 50 mg/kg or 75 mg/kg 5-FU, or PBS.Mice were injected with control (Ctrl) or STING-KO MC38 cells, and subjected to treatments, with each dose consisting of 25 mg/kg, 50 mg/kg or 75 mg/kg 5-FU, or PBS. B. Tumor volumes were quantified at the indicated days post cancer cell injection. N=5. Mice were injected with control (Ctrl) or STING-KO MC38 cells, and subjected to treatments, with each dose consisting of 25 mg/kg, 50 mg/kg or 75 mg/kg 5-FU, or PBS. C. Tumor and spleen weights at the endpoint for (B), with each dot representing a mouse.
Figure 3(A - D) APP endocytic trafficking followed in neurons expressing APP-RFP treated with siCD2AP or siControl using a pulse-chase assay with 22C11, analysed by epifluorescence microscopy.(A) Endocytosed APP detected with 22C11 (10 min pulse; top panels) and APP-RFP (bottom panels). Arrowheads identify axons (Ax) and dendrites (Dd) magnified on the right. Scale bars, 10 µm.(B) The amount of endocytosed APP (10 min) in cell body, dendrites and axons normalized to APP-RFP expression in the cell body quantified as percentage of siControl (n=3, NsiControl=19, NsiCD2AP=20; mean ± SEM).(C) Endocytosed APP detected with 22C11 (10 min pulse, 60 min chase; top panels) and APP-RFP (bottom panels). Arrowheads identify axons (Ax) and dendrites (Dd) magnified on the right. Scale bars, 10 µm.(D) The amount of endocytosed APP (60 min chase) was quantified as in (b) (n=3, NsiControl=23, NsiCD2AP=19; **PCB=0.001 siCD2AP vs. siControl, ***PDd=0.0004 siCD2AP vs. siControl, t-test; #P=0.0347 Dendrite vs. Cell body, ###P=0.0008 Dendrite vs. Axon, 1-way ANOVA with Tukey's multiple comparisons test; mean ± SEM). (E) Degradation of surface biotinylated APP (biotin APP) (time 0) chased for 20 and 60 min in neurons treated with siCD2AP, siBin1 or siControl. Biotin APP and total APP were detected with anti-APP (Y188) by western blot.(F) Quantification of biotinylated APP normalized to levels at time 0 (n=3-4, *P60min=0.0198, t-test; replicates and mean). (G and H) CD2AP (green) and APP (magenta) localisation in dendrites (G) and axons (I) of neurons expressing APP-RFP immunolabelled at 11 DIV with anti-CD2AP, analysed by spinning-disk confocal microscopy. Scale bars, 10 µm. The white squares are magnified on the right. Scale bars, 1 µm.(I) Quantification of colocalisation between CD2AP and APP in axons and dendrites (n=3-4, NDd=19, NAx=17; ****P<0.0001, t-test, mean ± SEM). (J and K) CD2AP (green) and Rab5 (magenta) localisation in dendrites (J) and axons (K) of neurons expressing Rab5-GFP analysed as in (G and H). Scale bars, 10 µm. The white squares are magnified on the right. Scale bars, 1 µm.(L) Quantification of colocalisation between CD2AP and Rab5-positive endosomes in dendrites (Dd) and axons (Ax) (n=3, NDd=17, NAx=23, ****P<0.0001, t-test, mean ± SEM).
Figure 7(A) Expression levels of IDH1 K224Ac in human CRC (n=90) and CRN (n=90) tissue specimens, assessed by immunohistochemistry. CRC, colorectal carcinoma samples; CRN, matched adjacent non-cancerous colorectal tissue specimens. (B) Kaplan-Meier survival curves of CRC cases with elevated (n=49) and reduced (n=41) IDH1 K224Ac. CRC, colorectal carcinoma samples. The overall survival was analyzed by the log-rank test using the Kaplan-Meier method. (C) A working model illustrating regulation of the HIF1α-SRC axis by SIRT2-dependent IDH1 deacetylation.
Figure 6ParBF exchange between foci was measured by FRAP an from two-foci cell of DLT1215 carrying pJYB234 A- Representative images of a photobleached cell during a FRAP experiment. The 488 nm laser was pulsed (Bleach) on one of the two foci at ~2.4 sec (black arrow). Red and blue arrows correspond to the bleached and unbleached focus, respectively. Time is indicated in second (upper right). The cell outline is drawn in red. Scale bar: 1 μm ParBF exchange between foci was measured by FRAP and FLIM (fluorescence lifetime imaging microscopy) from two-foci cell of DLT1215 carrying pJYB234 B- Quantification of ParBF-mVenus fluorescence intensity over time. The dynamics of fluorescence intensity is shown from averaging 18 independent measurements of the bleached (FRAP, red line) and unbleached (FLIM, blue line) foci. Foci fluorescence intensity in each experiment was normalized to the average intensity of each focus before photobleaching
is expressed in the developing cerebral cortex and neural stem cells (a) Immunostaining of TCF20, NESTIN, and merged with DAPI in E13 and E16 embryonic brain sections.Figure 1(a) Immunostaining of TCF20, NESTIN, and merged with DAPI in E13 and E16 embryonic brain sections. TCF20 is widely expressed in the brain. Data information: Scale bar in (A) e13, 50 μm, e16 100 μm (b) Costaining with the NSC markers NESTIN, SOX2, and PAX6 reveals that TCF20 is expressed in NSCs. (c) Absolute quantitative PCR of TCF20 in different developmental stages from E12 to P0. n=4 independently repeated tests. (d) Western blotting results show the changes in TCF20 protein expression level during brain development. (e) Statistical analysis of normalized band intensity in the western blot results. n= 4 samples for each group. (f) In utero electroporation of TCF20 shRNAs in the E13 cerebral cortex led to abnormal cell distribution. These electroporated embryos were collected at E16. (g) Statistical analysis of the distributions of GFP-positive cells in different brain regions. The percentage of GFP-positive cells in the CP (cortical plate), IZ (intermediate zone), and VZ/SVZ (ventricle zone and subventricle zone) was analyzed. n=7 brains. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.01(**), p<0.001(***). Scale bar in (F) 50 μm.
Figure 4Control and patient (index case, IC PSMC3) fibroblasts were exposed to a 16-h treatment with 30 nM of the proteasome inhibitor carfilzomib or left untreated (as a negative control). Following treatment, cells were collected and subjected to RIPA-mediated protein extraction prior to SDS-PAGE and subsequent western-blotting using antibodies specific for ubiquitin, TCF11/Nrf1, Rpt1 (PSMC2), Rpt3 (PSMC4), Rpt5 (PSMC3), Rpt6 (PSMC5), β1, β2, β5, β5i, β1i, PA28-α and α-Tubulin (loading control) as indicated. For the TCF11/Nrf1 staining, two exposure times are shown.
a) Colocalization of lysosomes (Lysotracker Green) with phagophores (mCherry-ULK1 and mCherry-ATG5 puncta) in glutamine-deprived U2OS cells.f3(a) Colocalization of lysosomes (Lysotracker Green) with phagophores (mCherry-ULK1 and mCherry-glutamine puncta) in glutamine-deprived U2OS cells. Scale bar, 25 μm. (b) Colocalization of LAMP1-positive lysosomes with GFP-LC3-labelled autophagosomes after glutamine deprivation in the absence or presence of 400 nM BafA1 in U2OS cells. Scale bar, 15 μm. (c) Immunofluorescence images of U2OS cells stained for p62 expression and LAMP1 following glutamine deprivation and BafA1 treatment. Nuclei were stained with DAPI. Scale bar, 15 μm. (d) Immunofluorescence images of cells stained with LAMP1 (lysosomes) and the Golgi marker, RCAS1, following glutamine deprivation. Nuclei were stained with DAPI. Scale bar, 15 μm. (e) U2OS cells were treated for 24 h with cell culture medium containing the indicated amino acids. Cell lysates were immunoblotted to detect changes in S6K phosphorylation and p62. ( (f) Immunofluorescence staining for LAMP1 (green) and mTOR (red) in U2OS cells subjected to glutamine deprivation (24 h), in the presence of 100 nM WYE-125132 (WYE-132). −AA denotes cells deprived of all amino acids. Scale bar, 15 μm. Error bars in all figures represent s.d. of three biological replicates. Ess., essential.
Deficiency in ciliogenesis by depletion of lamin A/C or nesprin 2 may be caused by disruption of the actin homeostasis.Figure 6A,B RPE cells expressing shRNAs specific to lamin A/C or nesprin 2 (shNesprin 2, clones #1 and #2) were stained for G-actin with DNase I (green). Representative images are shown (A). Scale bars, 20 μm. The proportion of nuclear G-actin fluorescence intensity to the whole cell was measured (B, n ≥ 67). C An equal amount of whole cell lysates from RPE cells (control) and those expressing shRNAs to importin 9 (shImportin 9, clones #1 and #2) was analyzed by immunoblotting with antibodies as indicated. D-F The cells expressing shRNAs to importin 9 were stained for G-actin (green) and F-actin (red). The representative images are shown (D). Scale bars, 20 μm or 10 μm (insets). The proportion of nuclear G-actin intensity to the whole cell (E, n > 152) and the cell spreading area (F, n ≥ 139) were measured. G-I RPE cells and those expressing shRNAs to importin 9 were serum-starved for 32 h and then treated with (+) or without (-) Cyto D for another 16 h and then stained for acetylated tubulin (white), pericentrin (green) and F-actin (red). Representative images are shown (G). The left insets show the merged signals of acetylated tubulin and pericentrin. Scale bars, 20 μm or 2 μm (magnified images). The percentage of the cells with cilia (H, n ≥ 385) and the length of cilia (I, n ≥ 111) were measured.
A) AUT2 is identical to YNL223w.Figure 1(A) AUT2 is identical to YNL223w. Genomic fragments of complementing plasmids and constructed subclones are illustrated. X, XbaI; B, BclI; N, NruI; H, HindIII. (B) Protein sequence of Aut2p. (C) Aut2p shows weak similarities to part of tubulin α2 from maize ( Montoliu et al., 1990).
Dose-dependent increase of C.f1Dose-dependent increase of C. albicans (SC5314) aggregation after 4-h exposure (a) and filamentation after 24-h exposure (b) to different concentrations of IL-17A in RPMI-1640 liquid medium. Cells aliquots were imaged by microscopy and magnified in the insets. Also note the increased turbidity of cell suspension at 24 h. Scale bars, 100 μm. (c) Percentages of germination of C. albicans untreated (vehicle-treated, open circle) or treated with IL-17A at 1 (black triangle) or 10 ng ml−1 (black square) for 4 or 24 h. Data are mean±s.d. P 0.05, IL-17A-treated versus vehicle-treated cells, at 4 h; P 0.01 (1 ng) and P 0.001 (10 ng) at 24 h (one-way analysis of variance Bonferroni post-test) (n=3). (d,e) IL-17A promotes biofilm formation. SEM images of C. albicans SC5314 untreated (d) or exposed to 10 ng ml−1 IL-17A (e) for 4 h and subsequently cultured onto plastic discs for 24 h. Scale bars, 10 μm. (f) IL-17A increases resistance to nitrogen starvation. Cells were first grown in YPD medium overnight and then switched to nitrogen-free SLAD medium in the presence of 10 ng ml−1 IL-17A. Viability, expressed as a percentage of colony-forming units at zero time, was measured over a 15-day period. Cells were exposed to 0.1 (black circle), 1 (black triangle) or 10 ng ml−1 (black square) IL-17A or drug vehicle (open circle) at the beginning of the cultures or, in the case of 1 ng ml−1 (open triangle), also added every two other days. Data are mean±s.d. P-values ranged from 0.05 to 0.001 for all samples treated with 10 ng IL-17A; P0.05, on days 3 and 5 for samples treated 1 ng IL-17A every two other days (one-way analysis of variance Bonferroni post-test) (n=3). (g,l) C. albicans cells are metabolically active. Untreated (g-i) or IL-17A-treated (10 ng ml−1 in RPMI-1640 liquid media for 24 h) (j-l) cells were red after staining with FUN-1 (g,j), green with Con A (h,k) and yellow in the merged images (i,l). Metabolically active cells are shown in red, and cell wall polysaccharides are shown in green. Scale bars, 5 μm. Images from one representative experiment out of two are shown.
Figure 2A. Schematic outline of genome-scale CRISPR-Cas9 screen for genes whose KO suppresses MMC hypersensitivity of SCAI KO cells. LD80, 80% lethal dose; NGS, next generation sequencing. B. MaGECK analysis of sgRNA enrichment in CRISPR screen in (A) following MMC treatment (n=3 technical replicates; false discovery rate (FDR) of 0.2 indicated by dotted line). FA genes are highlighted in blue. C. GO term analysis of significantly enriched genes (FDR<0.2) in CRISPR-Cas9 screen in (A), using Reactome pathways from PANTHER16.0. D. Immunoblot analysis of FANCA siRNA knockdown efficiency in U2OS cells. E. Clonogenic survival of U2OS and U2OS/SCAI KO cells transfected with non-targeting control (CTRL) or FANCA siRNAs and subjected to indicated doses of MMC for 24 h (mean±SEM; n=3 independent experiments). F. U2OS and U2OS/SCAI KO cells transfected with indicated siRNAs for 48 h were treated or not with MMC (90 nM) for 1 h, fixed 24 h later and co-stained with RPA2 antibody and DAPI. RPA2 foci were quantified by QIBC (≥3000 cells analyzed per condition; mean±SD; n=3 independent experiments; *, p<0.05; ns, not significant, two-tailed paired t-test). G. As in (F), except that cells were stained with γH2AX antibody (≥3000 cells analyzed per condition; mean±SD; n=3 independent experiments; **, p<0.01; ns, not significant, two-tailed paired t-test). H. Immunoblot analysis of the indicated U2OS cell lines. I. Clonogenic survival of U2OS and U2OS/FANCA KO cells subjected to indicated doses of MMC for 24 h (mean±SEM; n=3 independent experiments). J. Clonogenic survival of U2OS, U2OS/SCAI KO, U2OS/FANCA KO, and U2OS/FANCA+SCAI DKO cells subjected to indicated doses of MMC for 24 h (mean±SEM; n=3 independent experiments). K. Immunoblot analysis of U2OS, U2OS/SCAI KO, U2OS/FANCA KO, and U2OS/FANCA SCAI DKO cell lines exposed or not to MMC as indicated.
Figure 2WT or STX17-knockout (STX17KO) HeLa cells were starved with or without the presence of bafilomycin A1 (Baf A1, 100 nM) for 2 h, and cell lysates were subjected to Western blot analysis of LC3B and p62.   Quantifications of LC3B-II levels normalized to β-actin from cells treated as in (A); Data shown as means ± SEM of LC3B-II and β-actin ratios, n = 3; *, p < 0.05 (one-way ANOVA).   WT, STX16KO, STX17KO or STX16/STX17 double KO (STX16/STX17DKO) HeLa cells were starved with or without the presence of Baf A1 (100 nM) for 2 h, and cell lysates were subjected to Western blot analysis of LC3B.   Quantifications of LC3B-II levels normalized to β-actin from (C); Data shown as means ± SEM of LC3B-II and β-actin ratios, n = 3; †, not significant; **, p < 0.01 (one-way ANOVA).   WT or STX16/STX17DKO HeLa cells were starved in EBSS for 2 h, and subjected to ultrastructural analysis of the autophagic vesicles (AV) with electron microscopy. AVi: initial autophagic vacuoles; AVd: degradative autophagic vacuoles; G: Golgi apparatus. Image acquisition and counting as in Methods. Scale bars, 1 μm and 0.5 µm (top sections).   Quantifications of autophagic vesicles in WT and STX16/STX17DKO HeLa cells treated as (E); Data shown as means ± SEM of AV profiles relative to cytoplasmic area; †, not significant; *, p < 0.05 (two-way ANOVA). AV profiles from 47 images of each samples were counted.
Fig.Figure 1(A) Immunofluorescence staining of Lin-Sca-1+cKit+ (LSK) cells using an antibody specific for phosphorylated STAT5 (pSTAT5) or total STAT5. Scale bars represent 5μm. Bar graphs showing the quantification of nuclear localization presented as mean fluorescence intensity (MFI). Data are shown as means ± SD of three independent experiments. (B) Diagram showing HPC7 cells self-renew in the presence of stem cell factor (SCF) and undergo megakaryocytic differentiation in response to thrombopoietin (TPO). (C) Western blot analysis of HPC7 cells grown in the presence of SCF demonstrates nuclear and cytoplasmic uSTAT5 in the absence pSTAT5. K562 cell lysates were used as a positive control for pSTAT5. (D) Western blot analysis of HPC7 cells after TPO stimulation (100ng/ml) at the indicated time points. (E) Western blot analysis was performed after immunoprecipitation of STAT5 from unstimulated and TPO stimulated cells using antibodies to total STAT5 or to pSTAT5 (pY694/p699). 1% (5μg) of lysate was loaded as input control.