Journal: BMC Plant Biology

Article Title: Selenite activates the alternative oxidase pathway and alters primary metabolism in Brassica napus roots: evidence of a mitochondrial stress response

doi: 10.1186/s12870-014-0259-6

Figure Lengend Snippet: The effects of selenite on the ubiquitin-proteasome pathway in root tissue. (a) Proteasome activity in roots of untreated plants and plants treated with 50 μM selenite for 1 and 3 d. Shown are the mean and SE in 5 different plants. Values represent fluorescence of proteasomally-released AMC at the each time interval. Data are the mean of three biological replicates and standard deviation. Lowercase letters represent a significant difference in activity at each time point ( p < 0.05). (b) The accumulation of high-molecular weight ubiquitinated proteins in the roots of B. napus from untreated plants and plants treated with 50 μM selenite for 1 and 3 days, and then supplemented in 0.1% DMSO with or without 100 μM MG132 in for 8 hours. 50 μg of protein were separated on an 8% SDS gel, and ubiquitinated proteins were detected using anti-ubiquitin antiserum. The immunoblot is representative of at least three biological experiments, and numbers below each blot represent the mean pixel intensity of all the immunoreactive bands relative to control on day 1. Asterisks indicate a significant difference in band intensity in selenite-treated plants compared to untreated plants (p < 0.05). L = ladder.

Article Snippet: Ubiquitinated proteins were detected on 8% gels containing 50 μg of protein per lane, which reacted against ubiquitin antiserum (Santa Cruz Biotechnology) as previously described [ ].

Techniques: Ubiquitin Proteomics, Activity Assay, Fluorescence, Standard Deviation, High Molecular Weight, SDS-Gel, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Binding of the Human Complement Regulators CFHR1 and Factor H by Streptococcal Collagen-like Protein 1 (Scl1) via Their Conserved C Termini Allows Control of the Complement Cascade at Multiple Levels *

doi: 10.1074/jbc.M110.143727

Figure Lengend Snippet: Binding of Scl proteins to Factor H protein family members. A, CFHR1, CFHR2, CFHR3, and CFHR4A, as well as Factor H and FHL-1, were immobilized, and binding of streptococcal proteins Scl1.6, Scl1.55, and Scl2.28 was followed using a Strep-Tag II-specific HRP-coupled antibody. Streptococcal proteins Scl1.6 and Scl1.55 bound to CFHR1 and Factor H but not to any other Factor H family protein. Scl2.28 bound to none of the tested proteins (***, p < 0.001). B, 10% normal human serum was added to immobilized Scl1.6, Scl1.55, or Scl2.28, and bound CFHR1 and Factor H derived from human plasma were detected by a monoclonal antibody specific for CFHR1 (JHD10) and by a polyclonal antiserum specific for Factor H SCR1–4. The human regulators bound to Scl1.6 and Scl1.55 but not to Scl2.28. Mean values from triplicate wells of a representative experiment ± S.D. are shown (total of three independent experiments; ***, p < 0.001).

Article Snippet: Bound proteins were detected with polyclonal Factor H antiserum (1:1000, Comptech) and a secondary horseradish peroxidase (HRP)-coupled anti-goat antibody (1:1000, Dako), each diluted in blocking buffer.

Techniques: Binding Assay, Strep-tag, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Binding of the Human Complement Regulators CFHR1 and Factor H by Streptococcal Collagen-like Protein 1 (Scl1) via Their Conserved C Termini Allows Control of the Complement Cascade at Multiple Levels *

doi: 10.1074/jbc.M110.143727

Figure Lengend Snippet: Dose-dependent binding of CFHR1 and Factor H to streptococcal Scl1.6 and Scl1.55. Purified CFHR1 (A) and Factor H (B) bound dose-dependently to immobilized Scl1.6 (black circles) and Scl1.55 (black squares). No binding was detected for Scl1.1 (white triangles) and Scl2.28 (white diamonds). Error bars indicate S.D. C, binding of CFHR1 and Factor H to immobilized Scl1.6 (black), Scl1.55 (gray), Scl1.1 (light gray), and Scl2.28 (black dotted/dashed dotted) was analyzed by surface plasmon resonance SPR. CFHR1 (solid lines) and Factor H (dashed lines) bound to Scl1.6 and Scl1.55 but not to Scl1.1 or to Scl2.28. A representative experiment of three is shown.

Article Snippet: Bound proteins were detected with polyclonal Factor H antiserum (1:1000, Comptech) and a secondary horseradish peroxidase (HRP)-coupled anti-goat antibody (1:1000, Dako), each diluted in blocking buffer.

Techniques: Binding Assay, Purification, SPR Assay

Journal: The Journal of Biological Chemistry

Article Title: Binding of the Human Complement Regulators CFHR1 and Factor H by Streptococcal Collagen-like Protein 1 (Scl1) via Their Conserved C Termini Allows Control of the Complement Cascade at Multiple Levels *

doi: 10.1074/jbc.M110.143727

Figure Lengend Snippet: S. pyogenes strains used in this study

Article Snippet: Bound proteins were detected with polyclonal Factor H antiserum (1:1000, Comptech) and a secondary horseradish peroxidase (HRP)-coupled anti-goat antibody (1:1000, Dako), each diluted in blocking buffer.

Techniques: Binding Assay, Purification

Journal: The Journal of Biological Chemistry

Article Title: Binding of the Human Complement Regulators CFHR1 and Factor H by Streptococcal Collagen-like Protein 1 (Scl1) via Their Conserved C Termini Allows Control of the Complement Cascade at Multiple Levels *

doi: 10.1074/jbc.M110.143727

Figure Lengend Snippet: Binding of CFHR1 and Factor H, and of deletion fragments to Scl1.6 and Scl1.55. A, binding of Scl1.6, Scl1.55, and Scl2.28 to CFHR1 and to the two CFHR1 fragments, SCR1–2 and SCR3–5, was assayed. Scl1.6 and Scl1.55, but not Scl2.28, bound to CFHR1 and the C-terminal CFHR1 fragment SCR3–5. No Scl protein bound to the N-terminal CFHR1 fragment SCR1–2. Mean values from triplicate wells of a representative experiment ± S.D. are shown (total of three independent experiments). The insert shows the schematic domain structure of CFHR1 and that of the two fragments (***, p < 0.001). B, binding of CFHR1 as well as CFHR1 fragments to immobilized Scl1.6 (light gray) and Scl1.55 (dark gray) was assayed by SPR. CFHR1 (solid line) and the C-terminal fragment SCR3–5 (dashed-dotted line) bound to Scl1.6 and Scl1.55. The N-terminal CFHR1 SCR1–2 fragment (dotted line) did not bind to Scl1.6 and Scl1.55. C, binding of Scl1.6, Scl1.55, and Scl2.28 to Factor H, and the indicated immobilized Factor H fragments was assayed by ELISA. Scl1.6 and Scl1.55 bound to full-length Factor H as well as the C-terminal Factor H fragments SCR15–20 and SCR19–20. Scl2.28 did not bind to any Factor H fragment tested. Mean values from triplicate wells of a representative experiment ± S.D. are shown (total of three independent experiments). The insert shows the schematic domain structure of Factor H and that of the various fragments (***, p < 0.001). D, binding was also analyzed by SPR. Again, the C-terminal Factor H SCR15–20 (dashed line) bound to immobilized Scl1.6 (light gray) and Scl1.55 (dark gray). The Factor H SCR15–18 fragment (dotted line) did not bind.

Article Snippet: Bound proteins were detected with polyclonal Factor H antiserum (1:1000, Comptech) and a secondary horseradish peroxidase (HRP)-coupled anti-goat antibody (1:1000, Dako), each diluted in blocking buffer.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Binding of the Human Complement Regulators CFHR1 and Factor H by Streptococcal Collagen-like Protein 1 (Scl1) via Their Conserved C Termini Allows Control of the Complement Cascade at Multiple Levels *

doi: 10.1074/jbc.M110.143727

Figure Lengend Snippet: Identification of a linear Scl1.6-binding motif in SCR4 of CFHR1. A, linear peptides representing SCR4–5 of CFHR1 (residues 202–330), each with a length of 13 amino acids and an overlap of 10 residues, were spotted onto a membrane, and binding of Scl1.6 (upper panel) and Scl2.28 (lower panel) was followed. Scl1.6 bound to five spots representing the core motif, 232SVEYQ236 (upper panel). The nonbinding Scl2.28 did not bind to any peptide (lower panel). SCR4 of CFHR1 and SCR19 of the Factor H protein share sequence identity, and thus the binding motif is also contained in SCR19 (1133SVEYQ1137) of Factor H. B, structure of SCR4 and SCR5 of CFHR1 (corresponding to SCR19 and SCR20 of Factor H). The residues that form the linear binding motif are shown in black and are surface-exposed (Protein Data Bank code: 2G7I).

Article Snippet: Bound proteins were detected with polyclonal Factor H antiserum (1:1000, Comptech) and a secondary horseradish peroxidase (HRP)-coupled anti-goat antibody (1:1000, Dako), each diluted in blocking buffer.

Techniques: Binding Assay, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Binding of the Human Complement Regulators CFHR1 and Factor H by Streptococcal Collagen-like Protein 1 (Scl1) via Their Conserved C Termini Allows Control of the Complement Cascade at Multiple Levels *

doi: 10.1074/jbc.M110.143727

Figure Lengend Snippet: Ionic strength (NaCl) and heparin affect CFHR1 and Factor H binding to Scl1.6 and Scl1.55. A, the influence of ionic strength on CFHR1 binding to immobilized Scl1.6 and Scl1.55 was analyzed by increasing the NaCl concentration. CFHR1 bound to immobilized Scl proteins in the absence of NaCl, and NaCl reduced binding in a dose-dependent manner. B, similarly, NaCl affected the interaction of Factor H with Scl1.6 and Scl1.55. The arrows (A and B) indicate the physiological NaCl concentration. C, increasing concentrations of heparin dose-dependently affected the binding of CFHR1 to immobilized Scl1.6 and Scl1.55. D, similarly, heparin affected the binding of Factor H to immobilized streptococcal Scl1.6 and Scl1.55 proteins. Again, the effect was dose-dependent. The mean values derived from at least three separate experiments ± S.D. are shown. *, p < 0.05; **, p < 0.01; ***, p < 0.001; versus 0 mm NaCl or 0 μg/ml heparin.

Article Snippet: Bound proteins were detected with polyclonal Factor H antiserum (1:1000, Comptech) and a secondary horseradish peroxidase (HRP)-coupled anti-goat antibody (1:1000, Dako), each diluted in blocking buffer.

Techniques: Binding Assay, Concentration Assay, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Binding of the Human Complement Regulators CFHR1 and Factor H by Streptococcal Collagen-like Protein 1 (Scl1) via Their Conserved C Termini Allows Control of the Complement Cascade at Multiple Levels *

doi: 10.1074/jbc.M110.143727

Figure Lengend Snippet: Mutations in SCR20 and also disease-associated sequence variants affect Factor H binding to Scl1.6 and Scl1.55. Factor H SCR8–20, as well as four fragments that include aHUS-associated mutations (R1210C, V1197A, R1215G, and W1183V), and Factor H SCR15–20, which has heparin-binding residues exchanged (HepG), were attached via an immobilized monoclonal antibody (equal immobilization was verified in a parallel approach). Binding of Scl1.6, Scl1.55, and Scl2.28 applied to the fluid phase was detected using a Strep-Tag II-specific HRP-coupled antibody. Scl1.6 and Scl1.55, but not Scl2.28, bound to the unmodified fragment Factor H SCR8–20 and to Factor H SCR15–20, but the streptococcal proteins did not bind to any of the mutant proteins. Scl1.6 bound weakly to the Factor H SCR8–20 R1210C mutant construct. The mean values from triplicate wells of a representative experiment ± S.D. are shown (total of three independent experiments, ***, p < 0.001 versus no mutated proteins). The insert shows the structural model of SCR19–20 of Factor H (Protein Data Bank code: 2G7I). The positions of the five exchanged amino acids are indicated for the HepG mutant (upper panel). The disease-associated amino acids that were exchanged individually in four different aHUS cases are shown in black (lower panel). The linear Scl-binding motif is marked in dark gray.

Article Snippet: Bound proteins were detected with polyclonal Factor H antiserum (1:1000, Comptech) and a secondary horseradish peroxidase (HRP)-coupled anti-goat antibody (1:1000, Dako), each diluted in blocking buffer.

Techniques: Sequencing, Binding Assay, Strep-tag, Mutagenesis, Construct

Journal: The Journal of Biological Chemistry

Article Title: Binding of the Human Complement Regulators CFHR1 and Factor H by Streptococcal Collagen-like Protein 1 (Scl1) via Their Conserved C Termini Allows Control of the Complement Cascade at Multiple Levels *

doi: 10.1074/jbc.M110.143727

Figure Lengend Snippet: CFHR1 and Factor H bind to various S. pyogenes serotypes. A and B, binding of CFHR1, Factor H SCR15–20, or Factor H SCR15–18 to S. pyogenes M1-, M6-, M28- and M55-type, which express Scl1.1, Scl1.6, Scl2.28, and Scl1.55, respectively, was assayed by flow cytometry (A), and the MFI are shown (B). CFHR1 and Factor H SCR15–20 bound to the Scl1.55-expressing M55-type and to the Scl1.6-expressing M6-type bacteria. Both proteins also bound with weaker intensity to the M28-type streptococci, which express the non-CFHR1/C-terminal Factor H-binding Scl1.28 and Scl2.28 surface proteins. The two human proteins did not bind to M1-type streptococci. Factor H SCR15–18 did not bind to any of the tested streptococcal serotypes. One representative experiment of five independent experiments is shown. C, binding of CFHR1 and of Factor H fragments to immobilized S. pyogenes serotypes M1, M6, M28, and M55 was analyzed by whole-cell ELISA. Bound proteins were identified with a polyclonal antiserum that detects CFHR1 and Factor H. The C-terminal CFHR1 SCR3–5, but not the N-terminal CFHR1 SCR1–2 construct, bound to all four streptococcal serotypes, however with different intensities. Factor H construct SCR15–20 also bound, whereas construct SCR15–18 did not bind. The background intensities were subtracted to allow direct comparison of the four serotypes. The mean values derived from at least three separate experiments ± S.D. are shown (***, p < 0.001).

Article Snippet: Bound proteins were detected with polyclonal Factor H antiserum (1:1000, Comptech) and a secondary horseradish peroxidase (HRP)-coupled anti-goat antibody (1:1000, Dako), each diluted in blocking buffer.

Techniques: Binding Assay, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Construct, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Binding of the Human Complement Regulators CFHR1 and Factor H by Streptococcal Collagen-like Protein 1 (Scl1) via Their Conserved C Termini Allows Control of the Complement Cascade at Multiple Levels *

doi: 10.1074/jbc.M110.143727

Figure Lengend Snippet: Factor H binding to Scl proteins is influenced by CFHR1, CFHR1 competes with Factor H for binding, and CFHR1 affects cofactor activity of Scl-bound Factor H. A, Factor H bound to Scl1.6 and Scl1.55 (columns 1 and 2). CFHR1 competes with Factor H for binding, and increasing amounts of CFHR1 affected Factor H binding in a dose-dependent manner (columns 3–10). Bound Factor H was detected with a Factor H SCR1–4-specific antiserum. Mean values from triplicate wells of a representative experiment ± S.D. are shown (total of three independent experiments). ***, p < 0.001 versus column 1 and 2. B, Factor H bound to Scl1.6 and Scl1.55 retains complement regulatory activity. Factor H bound to the indicated Scl proteins; then C3b and Factor I was added, and after incubation the mixture was separated by SDS-PAGE and transferred to a membrane. Cofactor activity of bound Factor H was analyzed by assaying C3b cleavage products by Western blotting. C3b cleavage results in the appearance of the α′ 68-, α′ 43-, and α′ 41-kDa fragments for Factor H bound to Scl1.6 (lane 1) or Scl1.55 (lane 3). A control reaction with Factor H in the fluid phase was assayed in lane 7. A representative experiment of three is shown. C, CFHR1 affects Factor H-mediated complement control at the level of C3 convertase. Factor H, used at constant amounts together with increasing concentrations of CFHR1, bound to immobilized Scl1.6 or Scl1.55 at molar ratios of Factor H:CFHR1 1:0.5, 1:1, 1:2, 1:4, and 1:8 (lanes 3–7 and lanes 10–14). Following washing, C3b and Factor I were added, and after incubation the mixture was separated by SDS-PAGE and transferred to a membrane. Cofactor activity of bound Factor H was analyzed by assaying the appearance of C3b cleavage products by Western blotting using a polyclonal C3b antiserum. C3b cleavage is identified by the appearance of the α′ 68-, α′ 43-, and α′ 41-kDa fragments. CFHR1, by displacing Factor H, affects Factor H cofactor activity on the level of the C3 convertase. A representative experiment of three is shown.

Article Snippet: Bound proteins were detected with polyclonal Factor H antiserum (1:1000, Comptech) and a secondary horseradish peroxidase (HRP)-coupled anti-goat antibody (1:1000, Dako), each diluted in blocking buffer.

Techniques: Binding Assay, Activity Assay, Incubation, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Binding of the Human Complement Regulators CFHR1 and Factor H by Streptococcal Collagen-like Protein 1 (Scl1) via Their Conserved C Termini Allows Control of the Complement Cascade at Multiple Levels *

doi: 10.1074/jbc.M110.143727

Figure Lengend Snippet: CFHR1 bound to Scl1.6 and Scl1.55 inhibits TCC formation. A, the functional relevance of CFHR1·Scl1.6 and CFHR1·Scl1.55 complexes was analyzed in a hemolysis assay with sheep erythrocytes. TCC formation on sheep erythrocytes was induced with C5b6, C7, C8, and C9 and assayed by following erythrocyte lysis. Lysis was inhibited by increasing amounts of CFHR1·Scl1.6 and CFHR1·Scl1.55 complexes. Scl1.6 alone had a minor effect on hemolysis that was not amplified by increasing amounts. The mean values ± S.D. of three independent experiments are shown. B, TCC deposition was analyzed by the addition of TCC compounds C5b6, C7, C8, and C9 to CFHR1 bound to immobilized Scl1.6 and Scl1.55. For the negative control sample (no TCC), no C9 was added. TCC was detected using a TCC-specific antiserum. CFHR1 bound to Scl1.6 and Scl1.55 significantly reduced TCC deposition, whereas bound Factor H did not affect the TCC. Scl2.28, which bound to neither CFHR1 nor Factor H, did not influence TCC deposition. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Bound proteins were detected with polyclonal Factor H antiserum (1:1000, Comptech) and a secondary horseradish peroxidase (HRP)-coupled anti-goat antibody (1:1000, Dako), each diluted in blocking buffer.

Techniques: Functional Assay, Hemolysis Assay, Lysis, Amplification, Negative Control

Journal: Endocrinology

Article Title: Dual hormonal regulation of endocrine tissue mass and vasculature by adrenocorticotropin in the adrenal cortex.

doi: 10.1210/en.2004-0179

Figure Lengend Snippet: FIG. 5. Effect of Dex treatment on adrenal cortex vasculature and VEGF expression. Sections of adrenal glands from mice treated with 1 mg/kgd Dex for 0–14 d (D0 to D14) were immunostained for CD31 (A) or VEGF (B). The dotted lines indicate the border between adrenal cortex and medulla. Immunoreactivity is indicated by brown staining. Data from one representative experiment are shown. Similar results were obtained in three independent experiments.

Article Snippet: Slides were then incubated for 1 h with 0.5 g/ml rabbit polyclonal antihuman VEGF antiserum (A20; Santa Cruz Biotechnology, Santa Cruz, CA), which recognizes all VEGF isoforms or with a rat anti-BrdU at a dilution of 1/75 (clone BU1-75; Harlan, Indianapolis, IN).

Techniques: Expressing, Staining

Journal: Endocrinology

Article Title: Dual hormonal regulation of endocrine tissue mass and vasculature by adrenocorticotropin in the adrenal cortex.

doi: 10.1210/en.2004-0179

Figure Lengend Snippet: FIG. 6. Effects of Dex treatment on the expression of endocrine and endothelial cell markers. A, RT-PCR analysis of the expression of VEGF-A isoforms (the two bands correspond to specific amplification products of the VEGF120 and VEGF164 isoforms), VEGF-Rs (VEGF-R1/flt-1, VEGF-R2/flk-1, and neuropilin-1), endocrine cell markers (ACTH receptor/MC2-R, HDL receptor/SR-B1), and endothelial cell markers (PECAM and VE-cadherin) in the adrenal glands from mice treated with 1 mg/kgd Dex for 0–14 d (D0 to D14). RT-PCR amplification of HPRT mRNA was used as an internal standard for the normalization of the samples. The results from one representative experiment are shown. Similar results were obtained in three independent experiments. B and C, Quantitative determination of adrenal VEGF-A and VEGF-Rs mRNA levels by real-time RT-PCR was performed as described in Materials and Methods. The ratio of level of expression of the gene of interest to that of GAPDH was normalized to 1 in control adrenals. The relative quantities of the following mRNAs were plotted as a function of time of Dex treatment: VEGF-A (B); VEGF-R1 (C, solid line), VEGF-R2 (C, continuous dotted line), and neuropilin-1 (C, discontinuous dotted line). Each value represents the mean SEM from 10 to 15 mouse adrenals collected in three independent experiments.

Article Snippet: Slides were then incubated for 1 h with 0.5 g/ml rabbit polyclonal antihuman VEGF antiserum (A20; Santa Cruz Biotechnology, Santa Cruz, CA), which recognizes all VEGF isoforms or with a rat anti-BrdU at a dilution of 1/75 (clone BU1-75; Harlan, Indianapolis, IN).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Quantitative RT-PCR, Control

Journal: Iranian Journal of Basic Medical Sciences

Article Title: Protective role of alpha-lipoic acid against rhabdomyolysis-induced acute kidney injury in rats

doi: 10.22038/IJBMS.2024.74864.16252

Figure Lengend Snippet: Effect of glycerol 50% (10 ml/kg) and ALA (20 mg/kg) on TNF-α and NGAL levels rat in kidney tissues. A, C: Show immunoblot bands of the western blotting analysis and B, D: Display quantitative presentation of the immunoblots

Article Snippet: Next, they were incubated overnight (16–18 hr) at 4 °C on a rocker with rabbit polyclonal anti-serum against TNF-α antibody (Cell signaling, #3707, 1: 1,000), rabbit monoclonal anti-serum against NGAL (Abcam#216462, 1: 1,000) and mouse polyclonal anti-β-actin antibodies (Cell signaling #3700S, 1: 1,000).

Techniques: 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: 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