Journal: PLoS ONE

Article Title: Hsp20 Functions as a Novel Cardiokine in Promoting Angiogenesis via Activation of VEGFR2

doi: 10.1371/journal.pone.0032765

Figure Lengend Snippet: (A) The serum Hsp20 level was increased in response to in vivo 30 min-LAD occlusion followed by 24 h-reperfusion. Cardiac-specific overexpression of Hsp20 increased the Hsp20 concentration in the serum under basal and myocardial ischemia/reperfusion conditions. (n = 6; *, p<0.05 vs. WTs; #, p<0.05 vs. Sham groups). (B) The levels of Hsp20 in hearts from Hsp20-transgenic mice were determined by Western blot. α-Actin was used as an internal control (n = 4). (C) Myocardial ischemia/reperfusion stimulated the translocation of Hsp20 to the cardiomyocyte membrane, which was detected by fluorescence microscopy. Images are representative sections from four mice per group (green, Hsp20; red, α-Actin; Scale bar, 100 µm). (D) Quantitative data for expression of Hsp20 was evaluated using IPP 5.1 (n = 4; *, p<0.05 vs. WTs; #, p<0.05 vs. Sham groups).

Article Snippet: Samples or recombinant mouse Hsp20 protein (EIAab Science) were added and incubated for 2 h followed by three washes in PBST.

Techniques: In Vivo, Over Expression, Concentration Assay, Transgenic Assay, Western Blot, Control, Translocation Assay, Membrane, Fluorescence, Microscopy, Expressing

Journal: PLoS ONE

Article Title: Hsp20 Functions as a Novel Cardiokine in Promoting Angiogenesis via Activation of VEGFR2

doi: 10.1371/journal.pone.0032765

Figure Lengend Snippet: (A) Mild stress dose-dependently increased the amount of Hsp20 released from both Ad.GFP and Ad.Hsp20 infected myocytes, and (B) there was no difference in the release of lactate dehydrogenase (LDH), a marker of necrosis. Similar results were observed in two additional, independent experiments (*, p<0.05 vs. Ad.GFP-Control; #, p<0.05 vs. Ad.GFP).

Article Snippet: Samples or recombinant mouse Hsp20 protein (EIAab Science) were added and incubated for 2 h followed by three washes in PBST.

Techniques: Infection, Marker, Control

Journal: PLoS ONE

Article Title: Hsp20 Functions as a Novel Cardiokine in Promoting Angiogenesis via Activation of VEGFR2

doi: 10.1371/journal.pone.0032765

Figure Lengend Snippet: (A) Brefeldin A (BFA), which inhibits the classical protein transport pathway, did not block Hsp20 release into the media under either basal or hypoxia conditions (20 µM H 2 O 2 ). However, the release of Hsp20 from cardiomyocytes was reduced by both dimethyl amiloride (DMA), an exosome inhibitor, and Methyl-β-cyclodextrin (MBC), an inhibitor of lipid raft formation via depletion of membrane cholesterol. (B) The activity of acetylcholine esterase was used to quantify the amount of exosomes present in the media after various treatments. Similar results were observed in three additional, independent experiments (*, p<0.05 vs. Basal-Control; #, p<0.05 vs. H 2 O 2 -Control).

Article Snippet: Samples or recombinant mouse Hsp20 protein (EIAab Science) were added and incubated for 2 h followed by three washes in PBST.

Techniques: Blocking Assay, Membrane, Activity Assay, Control

Journal: PLoS ONE

Article Title: Hsp20 Functions as a Novel Cardiokine in Promoting Angiogenesis via Activation of VEGFR2

doi: 10.1371/journal.pone.0032765

Figure Lengend Snippet: (A) Recombinant human Hsp20 protein was added to HUVECs at various doses (80–2000 ng/ml) for 24 h. BSA was used as a control. Cell proliferation was determined by MTS. (B) Time-course effects of the Hsp20 protein(1000 ng/ml) on the HUVEC proliferation. (C) Representative photographs indicated the effects of recombinant human Hsp20 protein on the trans-well and tube formation of HUVECs. (D) Migration was quantified by counting cells that were moved through the membrane (Trans-well assay). (E) Tube formation was evaluated by the measurement of relative tube length. Similar results were observed in three additional, independent experiments (*, p<0.05 vs. Control).

Article Snippet: Samples or recombinant mouse Hsp20 protein (EIAab Science) were added and incubated for 2 h followed by three washes in PBST.

Techniques: Recombinant, Control, Migration, Membrane

Journal: PLoS ONE

Article Title: Hsp20 Functions as a Novel Cardiokine in Promoting Angiogenesis via Activation of VEGFR2

doi: 10.1371/journal.pone.0032765

Figure Lengend Snippet: (A) VEGFR2 coated on a plate dose-dependently captured the Hsp20 protein, whereas BSA coated did not arrest the Hsp20 protein. (B) Recombinant human Hsp20 protein significantly induced the expression of VRGFR2 in HUVECs, and co-localized with VEGFR2 in the cell surface. Images are representative sections from 20 fields per group (green, Hsp20; red, VEGFR2). Scale bar, 25 µm. (C–E) Blockade of the VEGFR2 signaling by a VEGFR2 neutralizing antibody and CBO-P11 (a VEGFR inhibitor) suppressed the HUVEC migration (C and D) and tube formation (C and E). Similar results were observed in three additional, independent experiments (*, p<0.05 vs. Control). (F and G) Immunoblots determined the levels of Akt, p-Akt, ERK and p-ERK in Hsp20-treated HUVECs. IgG or VEGFR2 antibody was pre-treated 30 min prior to the addition of Hsp20. β-actin was used as an internal control (n = 4; *, p<0.05 vs. Basal; #, p<0.05 vs. IgG+Hsp20).

Article Snippet: Samples or recombinant mouse Hsp20 protein (EIAab Science) were added and incubated for 2 h followed by three washes in PBST.

Techniques: Recombinant, Expressing, Migration, Control, Western Blot

Journal: PLoS ONE

Article Title: Hsp20 Functions as a Novel Cardiokine in Promoting Angiogenesis via Activation of VEGFR2

doi: 10.1371/journal.pone.0032765

Figure Lengend Snippet: (A) Blood vessels were stained for CD31 (capillary density) in heart sections of WT and Hsp20 TG mice, and (B) their quantitative analysis. For quantification of positively stained vessels, five sections of each heart (n = 4 hearts per group) were analyzed by an investigator who was blinded with respect to samples. Blood vessels were detected at low magnification (×200). Images are representative sections from four mice per group (green, α-Actin; red, CD31). Scale bar, 50 µm.

Article Snippet: Samples or recombinant mouse Hsp20 protein (EIAab Science) were added and incubated for 2 h followed by three washes in PBST.

Techniques: Staining

Journal: PLoS ONE

Article Title: Hsp20 Functions as a Novel Cardiokine in Promoting Angiogenesis via Activation of VEGFR2

doi: 10.1371/journal.pone.0032765

Figure Lengend Snippet: Intracellular Hsp20 is released outside cardiomyocytes via exosomes, and then interacts with VEGFR2. Consequently, its downstream signaling pathways (i.e. Akt and ERK) are activated, which promote myocardial angiogenesis.

Article Snippet: Samples or recombinant mouse Hsp20 protein (EIAab Science) were added and incubated for 2 h followed by three washes in PBST.

Techniques: Protein-Protein interactions

Journal:

Article Title: Evidence for a unique expression of CD4 on murine vaginal CD4 + cells

doi: 10.1046/j.1365-2567.2000.00028.x

Figure Lengend Snippet: CD4 protein on vaginal lymphoid cells by flow cytometry. Lymph node cell (106) and collagenase-digested vaginal lymphoid-like cells (105) from CBA/J mice were labelled with biotinylated (plus cychrome [CYC]-conjugated streptavidin) RM-4.5 or H129.19 and phycoerythrin (PE)-conjugated RM-4.4 epitope-distinct anti-CD4 antibodies. RM-4.5 and H129.19 anti-CD4 antibodies recognize an epitope in domain 1 of the CD4 protein, whereas RM-4.4 antibodies recognize an epitope in domain 3. Flow cytometric dual staining of lymph node cells (a, c and e) or vaginal lymphoid cells (b, d and f) with RM-4.4 and RM-4.5 anti-CD4 antibodies (c and d) and RM-4.4 and H129.19 anti-CD4 antibodies (e and f) is shown together with isotype control antibodies (a,b). The percentage in each quadrant represents the fluorescent positive cells within the lymphoid-like cell limits. Data shown are a representative of two experiments using 10 mice per experiment.

Article Snippet: Antibodies Four purified or fluorochrome/biotin-conjugated antibodies specific for the mouse CD4 protein were employed (PharMingen Corp., San Diego, CA).

Techniques: Flow Cytometry, Staining

Journal:

Article Title: Evidence for a unique expression of CD4 on murine vaginal CD4 + cells

doi: 10.1046/j.1365-2567.2000.00028.x

Figure Lengend Snippet: Immunohistochemical analysis of CD4+ lymph node and vaginal lymphoid cells under non-denaturing conditions. Lymphoid cells isolated from whole lymph nodes or collagenase-treated vaginal tissue from CBA/J mice were incubated with purified RM-4.5 anti-CD4 antibodies or with anti-rat IgG isotype-matched antibodies at 10 µg/ml prior to cytocentrifugation onto slides, and fixation. (a) and (b) show staining of lymph node cells with anti-rat IgG isotype matched antibodies, and RM-4.5 anti-CD4 antibodies, respectively. (c) and (d) show staining of vaginal lymphoid cells with anti-rat IgG isotype-matched antibodies, and RM-4.5 anti-CD4 antibodies, respectively. Arrows on each frame show representative stained cells. The figure is representative of three experiments using different mice.

Article Snippet: Antibodies Four purified or fluorochrome/biotin-conjugated antibodies specific for the mouse CD4 protein were employed (PharMingen Corp., San Diego, CA).

Techniques: Immunohistochemical staining, Isolation, Incubation, Purification, Staining

Journal:

Article Title: Evidence for a unique expression of CD4 on murine vaginal CD4 + cells

doi: 10.1046/j.1365-2567.2000.00028.x

Figure Lengend Snippet: Immunohistochemical analysis of CD4+ lymph node and vaginal lymphoid cells under denaturing conditions. Lymph nodes cells or cells collected from collagenase-treated vaginal tissue from CBA/J mice were cytocentrifuged onto slides, fixed and stained with RM-4.5 anti-CD4 antibodies or anti-rat IgG isotype-matched antibodies. (a) and (b) show staining of lymph node cells with anti-rat IgG isotype-matched antibodies, and RM-4.5 anti-CD4 antibodies, respectively. (c) and (d) show staining of vaginal lymphoid cells with anti-rat IgG isotype-matched antibodies, and RM-4.5 anti-CD4 antibodies, respectively. Arrows show representative areas of stained cells. The figure is representative of three experiments using different mice.

Article Snippet: Antibodies Four purified or fluorochrome/biotin-conjugated antibodies specific for the mouse CD4 protein were employed (PharMingen Corp., San Diego, CA).

Techniques: Immunohistochemical staining, Staining

Journal:

Article Title: Evidence for a unique expression of CD4 on murine vaginal CD4 + cells

doi: 10.1046/j.1365-2567.2000.00028.x

Figure Lengend Snippet: RT-PCR analysis of vaginal T-cell surface marker expression. (a) RT-PCR of total RNA extracted from vaginal lymphoid cells (VL) (5 × 106), whole vaginal tissue (VAG), and lymph node tissue (LN) of CBA/J mice was performed using Taq DNA polymerase and primers designed to amplify CD3 (549 bp), CD8 (513 bp), CD4 (615 bp), CD4B 983 bp, TCR-β (268 bp) and TCR-δ (222 bp) chain constant regions of murine systemic T lymphocytes. Amplification with primers for GAPDH (239 bp) was used as an internal control. The figure is representative of at least five experiments using different mice. (b) Southern blot analysis of CD4 and CD4B amplification products derived from vaginal and lymph node tissue cDNA using a γ-32P-end-labelled probe internal of the predicted amplification product.

Article Snippet: Antibodies Four purified or fluorochrome/biotin-conjugated antibodies specific for the mouse CD4 protein were employed (PharMingen Corp., San Diego, CA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Marker, Expressing, Amplification, Southern Blot, Derivative Assay

Journal:

Article Title: Evidence for a unique expression of CD4 on murine vaginal CD4 + cells

doi: 10.1046/j.1365-2567.2000.00028.x

Figure Lengend Snippet: lack of CD4 mRNA expression in RM-4.5– RM-4.4+ CD4+ vaginal cells. (a) Vaginal lymphoid cells extracted from CBA/J mice by collagenase-digestion and further purified by Ficoll–Paque® density gradient centrifugation were dual labelled with RM-4.4 and RM-4.5 anti-CD4 antibodies. Those cells that stained positively with RM-4.4 but not RM-4.5 monoclonal antibodies (RM-4.5– RM-4.4+) were FACS sorted. The RM-4.5– RM-4.4+ CD4+ cell-sorted population is represented in the encircled gate of quadrant B4. HeLa cells (5 × 105) were added to the sorted cell population and pelleted. Total RNA was extracted from RM-4.5– RM-4.4+-plus HeLa cells or HeLa cells alone, reverse transcribed and subjected to PCR using a high-efficiency Taq DNA polymerase and primers designed to amplify CD3, CD4, CD4B, TCR β-, and TCR δ chain constant regions of murine systemic T lymphocytes (b). The CD4 and CD4B amplification products shown are from column-purified primary amplification products subjected to a second round of amplification. Amplification with primers for GAPDH was used as an internal control. The figure is representative of two experiments using 40 mice in each experiment.

Article Snippet: Antibodies Four purified or fluorochrome/biotin-conjugated antibodies specific for the mouse CD4 protein were employed (PharMingen Corp., San Diego, CA).

Techniques: Expressing, Purification, Gradient Centrifugation, Staining, Amplification

Journal:

Article Title: In Cultured Oligodendrocytes the A/B-type hnRNP CBF-A Accompanies MBP mRNA Bound to mRNA Trafficking Sequences

doi: 10.1091/mbc.E07-10-1083

Figure Lengend Snippet: CBF-A, hnRNP A2, hnRNP A3, and hnRNP U are part of the same multiprotein complex. (A) Specificity of the affinity-purified peptide specific polyclonal anti-CBF-A antibody. Total protein extracts from HeLa cells were resolved by SDS-PAGE, blotted, and stained with Coomassie Blue (lane 1), immunostained with the CBF-A preimmune serum (lane 2), or with the affinity-purified anti-CBF-A antibody (lane 3) and with antibody SAK22 recognizing both CBF-A isoforms p37 and p42 (lane 4). (B) Sucrose gradient analysis of CBF-A, hnRNP A2, and hnRNP A3 from HeLa nuclear extracts. Fractions were resolved by SDS/PAGE and analyzed on immunoblots with antibodies to CBF-A and hnRNP A2/A3. (C) Schematic representation of recombinant hnRNP A2 and A3 and CBF-A constructs. (D) Pulldown experiment using S-tagged hnRNP A2, S-tagged hnRNP A3, or GST-tagged CBF-A constructs. The beads were incubated with HeLa nuclear extracts. Bound proteins were resolved by SDS PAGE, revealed by Coomassie staining and (E) analyzed on immunoblots with antibodies to CBF-A, hnRNP A2 and A3, and hnRNP U.

Article Snippet: Cloning, Expression, and Protein Purification Full-length hnRNP A2 (forward primer 5′-GGAATTCTTAGCGACTGAGTCCGCGATG, reverse primer 5′-ATAAGAATGCGGCCGCTGAAGCTGTTCTGTTACCTCTG) and hnRNP A3 ( Ma et al. , 2002 ) were cloned in pGEM-T (Promega, Madison, WI) and subsequently in pET30a (+) for expression (Novagen, Madison, WI).

Techniques: Affinity Purification, SDS Page, Staining, Western Blot, Recombinant, Construct, Incubation

Journal:

Article Title: In Cultured Oligodendrocytes the A/B-type hnRNP CBF-A Accompanies MBP mRNA Bound to mRNA Trafficking Sequences

doi: 10.1091/mbc.E07-10-1083

Figure Lengend Snippet: CBF-A binds the MBP mRNA RTS. (A) Sequences of wild-type (wtRTS) and scrambled RTS (scrRTS) used in this study. (B) Biotinylated wtRTS and scrRTS were conjugated to streptavidin Sepharose. Beads were incubated with HeLa nuclear, cytoplasmic, and high-salt protein extracts. Bound proteins were resolved by SDS-PAGE, revealed with Coomassie, and analyzed on immunoblots with antibodies to CBF-A and hnRNP A2 and A3. (C) RTS-binding assays using 33P-labeled wtRTS and scrRTS sequences. To perform EMSA, wtRTS and scrRTS probes were incubated with purified CBF-A and hnRNP A2 and A3 without affinity tags or (D) in the presence (+) or absence (−) of a 25-fold excess of unlabeled competitor RNA oligonucleotides as indicated. (E) Tissue distribution of CBF-A, analyzed on immunoblots, and normalized to the steady-state expression of histone H3.

Article Snippet: Cloning, Expression, and Protein Purification Full-length hnRNP A2 (forward primer 5′-GGAATTCTTAGCGACTGAGTCCGCGATG, reverse primer 5′-ATAAGAATGCGGCCGCTGAAGCTGTTCTGTTACCTCTG) and hnRNP A3 ( Ma et al. , 2002 ) were cloned in pGEM-T (Promega, Madison, WI) and subsequently in pET30a (+) for expression (Novagen, Madison, WI).

Techniques: Incubation, SDS Page, Western Blot, Binding Assay, Labeling, Purification, Expressing

Journal:

Article Title: In Cultured Oligodendrocytes the A/B-type hnRNP CBF-A Accompanies MBP mRNA Bound to mRNA Trafficking Sequences

doi: 10.1091/mbc.E07-10-1083

Figure Lengend Snippet: In cultured oligodendrocytes, CBF-A exhibits a granular cytoplasmic distribution which correlates with transported MBP mRNA. (A) Endogenous CBF-A (A–D and E–H) or (hnRNP A2 I–L and M–P) and MBP mRNA were simultaneously monitored by immuno-FISH and confocal microscopy. In D, arrows identify sites in which the distribution of CBF-A correlates with MBP RTS along processes. In E–H and M–P, oligodendrocyte processes are shown at approximately fivefold higher magnification. In H, arrowheads identify examples of CBF-A and MBP RTS-positive granules. In P, arrows point to examples of hnRNP A2- and MBP mRNA-positive granules. Scale bar, 20 μm. (B) Unbiased statistical quantification of individual CBF-A and MBP RTS-positive granules and (C) hnRNP A2 and MBP RTS-positive granules based on the immuno-FISH analysis. In both cases a linear correlation between the fluorescence intensity levels of CBF-A and RTS or hnRNP A2 and RTS is revealed.

Article Snippet: Cloning, Expression, and Protein Purification Full-length hnRNP A2 (forward primer 5′-GGAATTCTTAGCGACTGAGTCCGCGATG, reverse primer 5′-ATAAGAATGCGGCCGCTGAAGCTGTTCTGTTACCTCTG) and hnRNP A3 ( Ma et al. , 2002 ) were cloned in pGEM-T (Promega, Madison, WI) and subsequently in pET30a (+) for expression (Novagen, Madison, WI).

Techniques: Cell Culture, Confocal Microscopy, Fluorescence

Journal:

Article Title: In Cultured Oligodendrocytes the A/B-type hnRNP CBF-A Accompanies MBP mRNA Bound to mRNA Trafficking Sequences

doi: 10.1091/mbc.E07-10-1083

Figure Lengend Snippet: In oli-neu cells, the distribution of endogenous CBF-A correlates with hnRNP A2. (A and E) DAPI staining, (B and F) oli-neu cells stained with a mAb to hnRNP A2. (C and G) Oli-neu cells stained with the rabbit polyclonal peptide-specific anti-CBF-A antibody and (D and H) merged images. Scale bar, 20 μm. (B) Statistical quantification of CBF-A and hnRNP A2-positive granules based on the double immunofluorescence analysis and confocal microscopy in A. A linear correlation between the fluorescence signals of CBF-A and hnRNP A2 is revealed.

Article Snippet: Cloning, Expression, and Protein Purification Full-length hnRNP A2 (forward primer 5′-GGAATTCTTAGCGACTGAGTCCGCGATG, reverse primer 5′-ATAAGAATGCGGCCGCTGAAGCTGTTCTGTTACCTCTG) and hnRNP A3 ( Ma et al. , 2002 ) were cloned in pGEM-T (Promega, Madison, WI) and subsequently in pET30a (+) for expression (Novagen, Madison, WI).

Techniques: Staining, Immunofluorescence, Confocal Microscopy, Fluorescence

Journal:

Article Title: In Cultured Oligodendrocytes the A/B-type hnRNP CBF-A Accompanies MBP mRNA Bound to mRNA Trafficking Sequences

doi: 10.1091/mbc.E07-10-1083

Figure Lengend Snippet: CBF-A is associated with MBP mRNA in differentiating oligodendrocytes. (A) A complex containing CBF-A and hnRNP A2 is coprecipitated with the anti-CBF-A antibody from total protein extracts (Input) prepared from differentiating oli-neu cells in an RNA-dependent manner. Where indicated, extracts were treated with RNase A before immunoprecipitation. Bound proteins were resolved by SDS-PAGE and analyzed on immunoblots with antibodies to CBF-A and hnRNP A2. (B) qRT-PCR was performed on reverse-transcribed cDNA derived from RNA extracts of differentiating oli-neu cells, immunoprecipitated by CBF-A. The anti-CBF-A antibody leads to enrichment of MBP mRNA, as assessed with MBP-specific primers. Mock experiments and IgG pulldowns revealed negligible RNA enrichment. Input samples were considered to be 100%; thus all samples were divided by the inputs mean value. Data are presented as average of three independent experiments. Error bars, SEM. Importantly, in each case the percentages of immunoprecipitated mRNA are relative to the total amount of each individual mRNA species (input) analyzed.

Article Snippet: Cloning, Expression, and Protein Purification Full-length hnRNP A2 (forward primer 5′-GGAATTCTTAGCGACTGAGTCCGCGATG, reverse primer 5′-ATAAGAATGCGGCCGCTGAAGCTGTTCTGTTACCTCTG) and hnRNP A3 ( Ma et al. , 2002 ) were cloned in pGEM-T (Promega, Madison, WI) and subsequently in pET30a (+) for expression (Novagen, Madison, WI).

Techniques: Immunoprecipitation, SDS Page, Western Blot, Quantitative RT-PCR, Reverse Transcription, Derivative Assay