Journal: The Journal of Biological Chemistry

Article Title: Structure and Evolution of the Archaeal Lipid Synthesis Enzyme sn -Glycerol-1-phosphate Dehydrogenase *

doi: 10.1074/jbc.M115.647461

Figure Lengend Snippet: Expanded gap version of Dali-lite pairwise structural alignment of MJ G1PDH (36). Selected members of the larger enzyme superfamily including GDHs (Protein Data Bank codes 3CE9 and 1JQ5), ADHs (Protein Data Bank codes 1RRM, 3JZD, 3BFJ, and 4FR2), and DHQSs (Protein Data Bank codes 3QBE, 1XAG, and 1UJN) were used in the alignment. Secondary structural elements and residue numbering correspond to G1PDH. Residues that coordinate metals are highlighted in blue. The coenzyme binding motif is in italics, whereas residues that interact with coenzyme (NADP(H)) and substrate (DHAP) with respect to MJ G1PDH are highlighted in yellow and green, respectively. Reported intersubunit contacts between monomers are in red. Uppercase lettering indicates structurally equivalent positions with G1PDH, whereas lowercase indicates insertions relative to G1PDH.

Article Snippet: Previous biochemical characterizations of archaeal G1PDHs have shown the enzyme to be multimeric ( 17 , 42 ). table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Organism Class Protein Data Bank code-monomer Z-score a r.m.s.d. b lali c %id d C. acetobutylicum ATCC 824 GDH 3CE9-A 39.1 2.0 310 34 Sinorhizobium meliloti GDH 3UHJ-A 31.7 2.6 305 21 Geobacillus stearothermophilus GDH 1JQ5-A 30.3 2.5 300 21 Serratia plymuthica A30 GDH 4MCA-A 30.1 2.7 302 21 Schizosaccharomyces pombe GDH 1TA9-B 29.7 2.7 306 20 T. maritima GDH 1KQ3-A 29.5 2.6 301 26 E. coli Lactaldehyde reductase 1RRM-A 27.2 3.1 301 16 Ralstonia eutropha Fe-ADH 3JZD-A 26.9 2.9 302 15 Rhizobium sp. MTP-10005 MR 3W5S-A 26.8 3.2 300 15 Klebsiella pneumoniae POR 3BFJ-A 26.7 3.1 302 18 Agrobacterium tumefaciens MR 3HL0-A 26.8 3.0 302 15 Zymomonas mobilis ADH 2 3OWO-A 26.4 3.2 302 16 O. oeni POR 4FR2-A 26.4 3.2 302 20 Corynebacterium glutamicum ADH IV 3IV7-A 26.0 3.1 303 16 T. maritima Butanol dehydrogenase 1VLJ-B 25.2 3.5 299 18 T. maritima Fe-ADH 1VHD-A 25.1 3.2 294 18 Geobacillus thermoglucosidasius ADH 3ZDR-A 24.2 3.2 299 16 Shewanella denitrificans Fe-ADH 3RF7-A 24.2 3.4 290 17 E. coli Hypothetical oxidoreductase YqhD 1OJ7-A 23.6 3.9 300 17 Actinidia chinensis DHQS 3ZOK-D 23.0 3.5 294 16 Mycobacterium tuberculosis DHQS 3QBE-A 22.9 2.9 287 14 Aspergillus nidulans DHQS 1NVB-B 22.3 3.3 290 17 Streptomyces hygroscopicus Cyclase 4P53-A 21.0 2.9 277 16 Staphylococcus aureus DHQS 1XAG-A 20.9 3.3 282 17 Bacillus circulans 2-Deoxy- scyllo -inosose synthase 2GRU-A 20.3 3.2 283 18 Staphylococcus aureus DHQS 1XAH-A 19.5 3.2 264 16 Vibrio cholerae DHQS 3OKF-A 19.4 3.4 280 18 T. thermophilus DHQS 1UJN-A 18.8 3.4 271 14 Helicobacter pylori DHQS 3CLH-A 17.6 3.4 255 21 Open in a separate window a A measure of the statistical significance of the result relative to an alignment of random structures. b Root mean square deviation of α-carbon atoms. c Number of aligned residues. d Sequence identity between the two chains.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Structure and Evolution of the Archaeal Lipid Synthesis Enzyme sn -Glycerol-1-phosphate Dehydrogenase *

doi: 10.1074/jbc.M115.647461

Figure Lengend Snippet: Top structural alignment hits from the Dali-based structural alignment of MJ G1PDH ( 36 ) MR, maleylacetate reductase; POR, 1,3-propanediol oxidoreductase.

Article Snippet: Previous biochemical characterizations of archaeal G1PDHs have shown the enzyme to be multimeric ( 17 , 42 ). table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Organism Class Protein Data Bank code-monomer Z-score a r.m.s.d. b lali c %id d C. acetobutylicum ATCC 824 GDH 3CE9-A 39.1 2.0 310 34 Sinorhizobium meliloti GDH 3UHJ-A 31.7 2.6 305 21 Geobacillus stearothermophilus GDH 1JQ5-A 30.3 2.5 300 21 Serratia plymuthica A30 GDH 4MCA-A 30.1 2.7 302 21 Schizosaccharomyces pombe GDH 1TA9-B 29.7 2.7 306 20 T. maritima GDH 1KQ3-A 29.5 2.6 301 26 E. coli Lactaldehyde reductase 1RRM-A 27.2 3.1 301 16 Ralstonia eutropha Fe-ADH 3JZD-A 26.9 2.9 302 15 Rhizobium sp. MTP-10005 MR 3W5S-A 26.8 3.2 300 15 Klebsiella pneumoniae POR 3BFJ-A 26.7 3.1 302 18 Agrobacterium tumefaciens MR 3HL0-A 26.8 3.0 302 15 Zymomonas mobilis ADH 2 3OWO-A 26.4 3.2 302 16 O. oeni POR 4FR2-A 26.4 3.2 302 20 Corynebacterium glutamicum ADH IV 3IV7-A 26.0 3.1 303 16 T. maritima Butanol dehydrogenase 1VLJ-B 25.2 3.5 299 18 T. maritima Fe-ADH 1VHD-A 25.1 3.2 294 18 Geobacillus thermoglucosidasius ADH 3ZDR-A 24.2 3.2 299 16 Shewanella denitrificans Fe-ADH 3RF7-A 24.2 3.4 290 17 E. coli Hypothetical oxidoreductase YqhD 1OJ7-A 23.6 3.9 300 17 Actinidia chinensis DHQS 3ZOK-D 23.0 3.5 294 16 Mycobacterium tuberculosis DHQS 3QBE-A 22.9 2.9 287 14 Aspergillus nidulans DHQS 1NVB-B 22.3 3.3 290 17 Streptomyces hygroscopicus Cyclase 4P53-A 21.0 2.9 277 16 Staphylococcus aureus DHQS 1XAG-A 20.9 3.3 282 17 Bacillus circulans 2-Deoxy- scyllo -inosose synthase 2GRU-A 20.3 3.2 283 18 Staphylococcus aureus DHQS 1XAH-A 19.5 3.2 264 16 Vibrio cholerae DHQS 3OKF-A 19.4 3.4 280 18 T. thermophilus DHQS 1UJN-A 18.8 3.4 271 14 Helicobacter pylori DHQS 3CLH-A 17.6 3.4 255 21 Open in a separate window a A measure of the statistical significance of the result relative to an alignment of random structures. b Root mean square deviation of α-carbon atoms. c Number of aligned residues. d Sequence identity between the two chains.

Techniques:

Journal: Cell Death & Disease

Article Title: Pyruvate kinase L/R links metabolism dysfunction to neuroendocrine differentiation of prostate cancer by ZBTB10 deficiency

doi: 10.1038/s41419-022-04694-z

Figure Lengend Snippet: A , B IHC staining of PKLR and ZBTB10 on consecutive sections of a PCa TMA (CA4) in two selected cases. Scale bars, 100 μm. C Correlation analysis of the intensities of PKLR and ZBTB10 of the PCa TMA ( n = 49). PKLR expression was negatively associated with ZBTB10-expressing PCa samples. R , correlation coefficient; p , two-tailed p value. Significance was determined by correlation XY analyses in GraphPad Prism. D , E Tumor grade association analysis using a Chi-squared test of the CA9 PCa TMA. Intensities of PKLR ( D ) and ZBTB10 ( E ) staining were semiquantitatively scored using the H-index as follows: negative, weakly positive, moderately positive, and strongly positive. p values were calculated by a Chi-squared test performed using SPSS statistical 18.0 software. p < 0.001. F Proposed model for ADT-induced PKLR drives hormone-refractory PCa. Hormone-sensitive PCa activates AR signaling by increasing ZBTB10 leading to increased binding to the PKLR regulatory sequence and mediation of its transcriptional suppression. ADT induced inactivation of the AR-ZBTB10 pathway, leading to an abundance of PKLR through ZBTB10 loss of function. Overexpression of PKLR may upregulate glucose metabolism and NED progression of PCa cells. Targeting PKLR by potential PKLR inhibitors may reduce the PKLR-driven glycolysis and NED of ADT-resistant PCa.

Article Snippet: To mimic ADT, cells were cultured in RPMI-1640 medium with 5% CSS (ThermoFisher, 12676-029)-containing medium for 48 h or treated with 10 μM MDV3100 (Selleckchem, S1250) under standard culture conditions for 48 h. The AR ligand was treated with 10 nM DHT (Sigma-Aldrich) for 24 h. The candidate PKLR inhibitors (vilanterol, saquinavir, fosinopril, and salmeterol) were purchased from MedChemExpress (HY-14300, HY-17007, HY-B0382, and HY-14302), and the concentrations of each candidate drug for cell viability were treated with 0, 1, 5, 10, 25, and 50 μM for 24 h.

Techniques: Immunohistochemistry, Expressing, Two Tailed Test, Staining, Software, Binding Assay, Sequencing, Over Expression

Journal: Developmental biology

Article Title: Identification of regulators of germ stem cell enwrapment by its niche in C. elegans

doi: 10.1016/j.ydbio.2017.06.019

Figure Lengend Snippet: Genes required for DTC plexus formation in the L3/L4 stage.

Article Snippet: Scale bar, 10 μm. table ft1 table-wrap mode="anchored" t5 caption a7 Gene Public Name (Reference) Sequence Name (Gene) CDS Description DTC Plexus Defect PercentC Defect Tissue-specific RNAi Site of Action Mitochondria and energy metabolism nuo-1 C09H10.3 NADH ubiquinone oxidoreductase 7/11 64% DTC let-754 C29E4.8 Adenylate kinase 4/16 25% DTC, GCs C16A3.5 C16A3.5 NADH:ubiquinone oxidoreductase 4/20 20% DTC rpom-1 Y105E8A.23 Mitochondrial RNA polymerase 4/20 20% dld-1 LLC1.3 Dihydrolipoamide dehydrogenase 3/19 16% GCs Transport and vesicle trafficking sec-10 C33H5.9 Exocyst subunit 7/18 39% DTC emo-1 F32D8.6 Sec61p gamma subunit 4/15 27% DTC, GCs copa-1 Y71F9AL.17 COPI complex alpha subunit 3/9 33% DTC, GCs sar-1 ZK180.4 SAR (Secretion Associated, Ras-related) COPII vesicle coat protein 3/19 16% dyn-1 C02C6.1 Dynamin GTPase 5/24 21% RNA Regulation rnp-7 K04G7.10 RNP (RRM RNA binding domain)-containing 4/20 20% DTC snr-2 W08E3.1 Small nuclear ribonucleoprotei n polypeptide 3/16 19% GCs Protein Synthesis and Degradation dre-1 K04A8.6 F-box domain-containing protein 9/29 31% GCs hars-1 T11G6.1 Histidyl tRNA synthetase 8/10 80% DTC rpl-11.1 T22F3.4 Large ribosomal subunit L11 5/17 29% F23B12.7 F23B12.7 Constituent of 66S pre-ribosomal particles 7/19 37% W07E6.2 W07E6.2 WD-repeat protein RSA4/Notchless 4/26 15% GCs ppp-1 C15F1.4 Translation initiation factor eIF2B gamma subunit 3/19 16% uba-1 C47E12.5 Ubiquitin-activating enzyme 6/18 33% GCs Chromatin and transcription lin-40 T27C4.4 Histone deacetylase complex, chromatin binding 9/19 47% DTC pab-1 Y106G6H.2 Poly(A)-binding protein 7/29 24% DTC, GCs his-72 Y49E10.6 H3 histone 3/20 15% GCs Cell signaling clr-1 F56D1.4 Receptor tyrosine phosphatase 5/21 24% sur-6 F26E4.1 Regulatory subunit of serine/ threonine protein phosphatase 2A 3/19 16% lin-3 F36H1.4 EGF family ligand 4/27 15% Nuclear Transport and signaling ran-1 K01G5.4 Ran GTPase 5/15 33% DTC, GCs ftt-2/par-5 a F52D10.3 14-3-3 signaling and scaffolding protein 5/18 28% DTC ( ftt-2 ), GCs ( par-5 ) Focal adhesion unc-112 C47E8.7 Fermitin, PH domain-containing protein 9/17 53% DTC Uncharacterized nematode-specific gene R08C7.1 R08C7.1 Protein coding gene 6/25 24% DTC, GCs Open in a separate window Genes are grouped by reported function or predicted function based on homologs.

Techniques: Sequencing, RNA Binding Assay, Ubiquitin Proteomics, Histone Deacetylase Assay, Binding Assay, Scaffolding

Journal: Frontiers in Zoology

Article Title: Proteins from toad’s parotoid macroglands: do they play a role in gland functioning and chemical defence?

doi: 10.1186/s12983-023-00499-8

Figure Lengend Snippet: Proteins identified in the extract from parotoids of the common toad Bufo bufo based on tandem mass spectrometry analysis

Article Snippet: P0C273 , 1019 , , , 14,949 , , , 23 , , , 31 , , , Ubiquitin 60S ribosomal protein L40 , , , DNA repair, cell cycle regulation, protein degradation, cell signalling , , , , , 1, 6, 7, 11.

Techniques: Mass Spectrometry, Sequencing, Binding Assay, Control, Activity Assay, Ubiquitin Proteomics, Migration, Transduction, Cell Differentiation, Phospho-proteomics, Coagulation, Membrane, Clinical Proteomics, Protein Binding

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: The SH2 domain of STAT5A is required for efficient binding to Src kinases. (A) Domain structure of fluorescently labeled STAT5A-eYFP. (B) Subcellular localization of STAT5A-eYFP in the absence or presence of Epo. HeLa T-REx HA-EpoR cells stably transfected with STAT5A-eYFP were stimulated with 1 U/ml Epo for 30 min and the localization of STAT5A-eYFP was analyzed by confocal microscopy. Scale bars: 20 μm. (C) Subcellular localization of STAT5A-eYFP (upper panel), STAT5A R618Q -eYFP (middle panel) and STAT3-eYFP (lower panel) was investigated in the presence of vSrc-dsRed. HeLa T-REx vSrc-dsRed cells were treated with 5 ng/ml doxycycline and transfected with the indicated constructs and the distribution of fluorescently labeled fusion proteins was analyzed after 24 h by confocal microscopy. Scale bars: 20 μm. (D) Quantification of the relative subcellular distribution of eYFP-labeled STAT3 and STAT5A constructs in HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP (B) and HeLa T-REx vSrc-dsRed cells transfected with STAT5A-eYFP, STAT5A R618Q -eYFP or STAT3-eYFP (C) . The expression of the HA-EpoR and vSrc-dsRed was induced with 5 ng/ml doxycycline for 24 hours. Mean fluorescence intensities (MFI) of the cytoplasm and nucleus were determined using the Zen 2012 software and changes in the ratio between the compartments were plotted. The data shown are means ± SD of n = 30 cells and were statistically evaluated by Student’s t -test. ***p < 0.0005. n.s. = not significant. (E + F) HeLa T-REx FRT cells were co-transfected with plasmids coding for STAT5A-eYFP or STAT5A R618Q -eYFP and vSrc-dsRed or Hck-dsRed. Fluorescently labeled STAT5 was immunoprecipitated from cell lysates using a GFP antibody and analyzed by immunoblotting for the presence of vSrc-dsRed or Hck-dsRed 24 h after transfection. The expression and phosphorylation of STAT5A and vSrc/Hck proteins was analyzed in the whole cellular lysates (WCL) using antibodies against pY 416 -Src, Src, Hck, pY 694/699 -STAT5A/B and GFP.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Binding Assay, Labeling, Stable Transfection, Transfection, Confocal Microscopy, Construct, Expressing, Fluorescence, Software, Immunoprecipitation, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: STAT5A binds to the phosphorylated activation loop of SFK. (A) Domain structure of vSrc-dsRed. Selected amino acids are highlighted. A multiple sequence alignment of the activation loop of SFK is shown. Autophosphorylation site is highlighted (red). (*) conserved amino acids, (:) similar properties . Bold characters highlight peptide sequence used for precipitation. (B + C) HeLa T-REx FRT cells stably expressing STAT5A-eYFP were transfected with the indicated vSrc-dsRed variants. Phosphorylation was analyzed 24 h after transfection using antibodies against pY 416 -Src, Src, pY 694/699 -STAT5A/B and STAT5A. CTRL = untransfected cells. (D) Quantification of relative subcellular distribution of STAT5A in HeLa T-REx FRT stably expressing STAT5A-eYFP and the indicated vSrc-dsRed mutants. Mean fluorescence intensity (MFI) of eYFP-fluorescence in the cytoplasm and nucleus were determined using the Zen 2012 software and changes in the ratio between the compartments were plotted. Data show means ± SD of n = 10 cells and were statistically evaluated by Student’s t -test. ***p < 0.0005, **p < 0.005, *p < 0.05, n.s. = not significant. (E) HeLa T-REx FRT cells stably expressing STAT5A-eYFP were transfected with vSrc K295N -dsRed or vSrc Y416F -dsRed. Subcellular distribution of STAT5A-eYFP was analyzed 24 h after transfection by confocal microscopy. Scale bars: 20 μm. (F + G) HeLa T-REx FRT cells were co-transfected with plasmids coding for vSrc-dsRed (Hck-dsRed), vSrc K295N -dsRed (Hck K269N -dsRed) or vSrc Y416F -dsRed (Hck Y390F -dsRed) and STAT5A-eYFP. STAT5-eYFP was immunoprecipitated from cell lysates using a GFP antibody and analyzed by immunoblotting for the presence of vSrc-dsRed (Hck-dsRed) 24 h after transfection. Expression and phosphorylation of STAT5A and vSrc proteins was analyzed in the WCL using antibodies against pY 416 -Src, Src, Hck, pY 694/699 -STAT5A/B and GFP. (s) short exposure, (l) long exposure. (H) HeLa T-REx FRT cells expressing STAT5A-eYFP or STAT5A R618Q -eYFP were lysed and incubated with a Src-peptide containing tyrosine- or phosphotyrosine 416. Precipitates and WCL were analyzed by immunoblotting using a GFP-specific antibody.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Activation Assay, Sequencing, Stable Transfection, Expressing, Transfection, Fluorescence, Software, Confocal Microscopy, Immunoprecipitation, Western Blot, Incubation

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: SFK-mediated cytoplasmic localization of STAT5A is dominant over BCR-ABL induced nuclear accumulation. (A) HeLa T-REx BCR-ABL cells were transiently transfected with STAT5A-eYFP and either treated with 5 ng/ml doxycycline for 24 h to induce BCR-ABL expression (lower panel) or left untreated (upper panel). Fixation was performed with methanol. Fixed cells were stained for BCR-ABL using a cABL-specific primary antibody and a secondary antibody conjugated to Alexa Fluor-405. The subcellular distribution of STAT5A-eYFP was analyzed by confocal microscopy. Scale bars: 20 μm. (B) The subcellular distribution of STAT5A-eYFP was investigated in the presence of vSrc-dsRed (upper panel), vSrc K295N -dsRed (middle panel) or vSrc Y416F -dsRed (lower panel) in HeLa T-REx BCR-ABL cells that were treated with 5 ng/ml doxycycline for 24 h. Fixation was performed with methanol. Fixed cells were stained for BCR-ABL using an Abl-specific primary antibody and a secondary antibody conjugated to Alexa Fluor-405. Scale bars: 20 μm. (C) HeLa T-REx BCR-ABL cells were co-transfected with vSrc-dsRed, or the respective kinase activity affecting mutants vSrc K295N -dsRed or vSrc Y416F -dsRed and STAT5A-eYFP. The cells were either treated with 5 ng/ml doxycycline for 24 h (lanes 1–3) to induce the expression of BCR-ABL or left untreated (lane 4). Protein expression and phosphorylation in the cellular extracts was investigated by immunoblotting with antibodies against pY 412 -cABL, cABL, pY 694/699 -STAT5A/B, STAT5A, pY 416 -Src and Src. α-Tubulin served as a loading control.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Transfection, Expressing, Staining, Confocal Microscopy, Activity Assay, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: Binding of STAT5A to Src kinases interferes with dimerization. (A) HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP were transfected with STAT5A-FLAG. The cells were treated with 5 ng/ml doxyxcyline for 24 h to induce the expression of the HA-tagged EpoR and stimulated with 5 U/ml Epo for 30 minutes or left untreated. HeLa T-REx vSrc-dsRed cells stably expressing STAT5A-eYFP were transfected with STAT5A-FLAG. The expression of vSrc-dsRed was induced for 8 h with 5 ng/ml doxycycline or the cells were left untreated. STAT5A-eYFP was immunoprecipitated from cell lysates using a GFP antibody and analyzed by immunoblotting for the presence of STAT5A-FLAG. The expression and phosphorylation of STAT5A-eYFP and STAT5A-FLAG was analyzed in the WCL using antibodies against pY 694/699 -STAT5A/B, GFP and the FLAG-tag. (B) HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP were treated with 5 ng/ml doxyxcyline for 24 h to induce the expression of the human EpoR and stimulated with 5 U/ml Epo for 30 minutes or left untreated. HeLa T-REx vSrc-dsRed cells stably expressing STAT5A-eYFP or a STAT5A S710F -eYFP were treated with 5 ng/ml doxyxcline for 8 h or the cells were left untreated. Cellular extracts were prepared under native conditions and STAT5A-eYFP dimers were separated from monomers by blue native PAGE electrophoresis (NP). STAT5A-eYFP dimer complexes were measured by the detection of the eYFP fluorescence. The cellular extracts were subjected to immunoblotting using antibodies against pY 694/699 -STAT5A/B, STAT5A, Src and the HA-tag of the EpoR. (C) Confocal microscopy analysis of HeLa T-REx FRT cells co-expressing vSrc-dsRed together with STAT5A S710F -eYFP (upper panel), a serine phosphorylation mimicking mutant STAT5A S710D -eYFP (middle panel) or a serine phosphorylation deficient mutant STAT5A S710A -eYFP (lower panel). Methanol fixation was performed 24 h after transfection. Scale bars: 20 μm.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Binding Assay, Stable Transfection, Expressing, Transfection, Immunoprecipitation, Western Blot, FLAG-tag, Blue Native PAGE, Electrophoresis, Fluorescence, Confocal Microscopy, Mutagenesis

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: Activated SFK interfere with dimerization and nuclear translocation of pSTAT5A in BCR-ABL expressing cells. Left scheme: Classical activation of the JAK2-STAT5A signaling pathway downstream of the EpoR. Right scheme: BCR-ABL directly phosphorylates STAT5A Y694 resulting in STAT5A dimerization, nuclear accumulation and finally target gene expression . In the presence of BCR-ABL, a predominantly cytoplasmic localization of pSTAT5A is achieved (i) upon binding to the scaffolding adaptor Gab2 resulting in pro-survival signaling through PI3K/Akt activation and (ii) through binding of the STAT5A SH2 domain to the phosphorylated activation loop of SFK, a mechanism that interferes with STAT5A dimerization and subsequent nuclear accumulation. Constitutively active STAT5A S710F escapes the SFK-mediated cytoplasmic retention. Flashes indicate phosphorylation events.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Translocation Assay, Expressing, Activation Assay, Binding Assay, Scaffolding

Journal: BMC Microbiology

Article Title: Antibiotics resistance and toxin profiles of Bacillus cereus -group isolates from fresh vegetables from German retail markets

doi: 10.1186/s12866-019-1632-2

Figure Lengend Snippet: Characteristics of whole genome datasets of selective B. cereus s.l. strains

Article Snippet: A homologous group filtering and a group alignment were performed by PATRIC pipeline [ ] and an estimated phylogenetic tree from concatenated alignment sequences was calculated within the type strains B. cereus ATCC 14579 T (DSM 31 T ), B. thuringiensis ATCC 10792 T (DSM 2046 T ), B. toyonensis BCT-7112 T (CECT 876 T ), B. weihenstephanensis WSBC 10204 T (DSM 11821 T ), B. mycoides ATCC 6462 T (DSM 2048 T ) and Bacillus pseudomycoides AFS069374 T using a FastTree method [ ].

Techniques:

Journal: BMC Microbiology

Article Title: Antibiotics resistance and toxin profiles of Bacillus cereus -group isolates from fresh vegetables from German retail markets

doi: 10.1186/s12866-019-1632-2

Figure Lengend Snippet: The amino acid sequences of the hblC gene of Bacillus cereus s.l. strains were clustered in this study. Most of strains reacted Hbl-positive with the Duopath test but two strains (B26 and MS17) were Hbl-negative. The phylogenetic analysis was carried out using the Jukes-Cantor model for genetic distance and the unweighted pair-group method using arithmetic averages (UPGMA) algorithm of the Geneious® program. The branch length and branch support values indicate the units of substitutions per site of the sequence alignment

Article Snippet: A homologous group filtering and a group alignment were performed by PATRIC pipeline [ ] and an estimated phylogenetic tree from concatenated alignment sequences was calculated within the type strains B. cereus ATCC 14579 T (DSM 31 T ), B. thuringiensis ATCC 10792 T (DSM 2046 T ), B. toyonensis BCT-7112 T (CECT 876 T ), B. weihenstephanensis WSBC 10204 T (DSM 11821 T ), B. mycoides ATCC 6462 T (DSM 2048 T ) and Bacillus pseudomycoides AFS069374 T using a FastTree method [ ].

Techniques: Sequencing

Journal: BMC Microbiology

Article Title: Antibiotics resistance and toxin profiles of Bacillus cereus -group isolates from fresh vegetables from German retail markets

doi: 10.1186/s12866-019-1632-2

Figure Lengend Snippet: Origin of Bacillus cereus -groupstrains investigated in this study

Article Snippet: A homologous group filtering and a group alignment were performed by PATRIC pipeline [ ] and an estimated phylogenetic tree from concatenated alignment sequences was calculated within the type strains B. cereus ATCC 14579 T (DSM 31 T ), B. thuringiensis ATCC 10792 T (DSM 2046 T ), B. toyonensis BCT-7112 T (CECT 876 T ), B. weihenstephanensis WSBC 10204 T (DSM 11821 T ), B. mycoides ATCC 6462 T (DSM 2048 T ) and Bacillus pseudomycoides AFS069374 T using a FastTree method [ ].

Techniques: Isolation