Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Biological Significance of Decreased HSP27 in Human Atherosclerosis

doi: 10.1161/01.atv.0000220108.97208.67

Figure Lengend Snippet: Figure 1. HSP27 proteolysis by plasmin in vitro and ex vivo. A, Recombinant HSP27 was incubated with 125 nmol plasmin for increas- ing periods of time (15 to 60 minutes) with or without inhibitors (VFK and aprotinin). B, Incubation of mammary endartery-released HSP27 with plasmin (125 mmol/L) for 18 hours led to its degradation and aggregation in the conditioned medium, and this effect is pre- vented in the presence of the plasmin inhibitor, aprotinin. C, Incubation of recombinant HSP27 with atherosclerotic plaques, led to HSP27 proteolysis in the conditioned medium. This effect is partially prevented by aprotinin, a serine-protease inhibitor. D, Coincuba- tion of mammary endarteries (M), known to release HSP27, with atherosclerotic plaque samples (P), as a source of proteases, led to HSP27 proteolysis in the conditioned medium. The empty dashed arrow indicates the band corresponding to HSP27 degradation prod- uct and the plain arrow shows the aggregated forms of HSP27.

Article Snippet: Human VSMCs were grown to 50% confluence in 12-well plates and transfected with a mixture composed of 2.5 mol/L CaCl2, 25 nmol small interfering RNA (siRNA) (human HSP27 siRNA sc-29350; Santa Cruz Biotechnology) and calcium phosphate buffer (280 mmol/L NaCl, 1.5 mmol/L Na2HPO4, 12 mmol/L glucose, 10 mmol/L KCl, 50 mmol/L Hepes, pH7) in 10% FCS complete medium.

Techniques: In Vitro, Ex Vivo, Recombinant, Incubation, Protease Inhibitor

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Biological Significance of Decreased HSP27 in Human Atherosclerosis

doi: 10.1161/01.atv.0000220108.97208.67

Figure Lengend Snippet: Figure 3. HSP27 levels modulate plasmin-induced VSMC anoikis. A, Human VSMCs were transfected with calcium phos- phate alone (control of transfection, CT) or with HSP27-siRNA (25 to 50 nM). HSP27 (upper panel) and HSP70 (lower panel) levels were analyzed by Western-blot analysis. Ccontrol cells. B, Immunofluorescence detection of HSP27 in VSMCs trans- fected or not with HSP27-siRNA (25 nM) for 24 hours and cul- tured for additional 48 hours. C, Representative microphoto- graphs of control (C) human VSMCs or treated with plasmin (Pn) for 18 hour in nontransfected cells (upper panel) or in cells transfected with 25 nmol HSP27-siRNA (lower panel).

Article Snippet: Human VSMCs were grown to 50% confluence in 12-well plates and transfected with a mixture composed of 2.5 mol/L CaCl2, 25 nmol small interfering RNA (siRNA) (human HSP27 siRNA sc-29350; Santa Cruz Biotechnology) and calcium phosphate buffer (280 mmol/L NaCl, 1.5 mmol/L Na2HPO4, 12 mmol/L glucose, 10 mmol/L KCl, 50 mmol/L Hepes, pH7) in 10% FCS complete medium.

Techniques: Transfection, Control, Western Blot

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Biological Significance of Decreased HSP27 in Human Atherosclerosis

doi: 10.1161/01.atv.0000220108.97208.67

Figure Lengend Snippet: Figure 4. Effect of HSP27 on apoptosis in vitro. A, Human VSMCs were incubated with plasmin (Pn) for 18 hours in VSMCs transfected or not with HSP27-siRNA (25 nmol). Viability was assessed by the MTT test, which quantifies the remaining adherent cells. Untreated control cells (C) represent 100% of viable adherent cells and the results are expressed as a per- centage of control (mean of 3 separate experiments SD, *P0.05 vs control; †P0.05 vs Pn). B, Quantification of DNA fragmentation in human VSMCs incubated with plasmin (Pn) for 18 hours transfected or not with HSP27-siRNA (25 nmol). Results are expressed in A405 nm 103 (meanSD, *P0.05 vs control; †P0.05 vs Pn). C, Detection of apoptosis in mammary arteries incubated with plasmin (Pn) for 24 hours. Apostain- positive nuclei were absent in control (C) mammary endarteries.

Article Snippet: Human VSMCs were grown to 50% confluence in 12-well plates and transfected with a mixture composed of 2.5 mol/L CaCl2, 25 nmol small interfering RNA (siRNA) (human HSP27 siRNA sc-29350; Santa Cruz Biotechnology) and calcium phosphate buffer (280 mmol/L NaCl, 1.5 mmol/L Na2HPO4, 12 mmol/L glucose, 10 mmol/L KCl, 50 mmol/L Hepes, pH7) in 10% FCS complete medium.

Techniques: In Vitro, Incubation, Transfection, Control

Journal: Annals of Laboratory Medicine

Article Title: In vitro Stability of Heat Shock Protein 27 in Serum and Plasma Under Different Pre-analytical Conditions: Implications for Large-Scale Clinical Studies

doi: 10.3343/alm.2016.36.4.353

Figure Lengend Snippet: Stability results for HSP27: effects of immediate and delayed processing of blood samples with storage at 4℃

Article Snippet: Circulating HSP27 concentrations were measured by using the Human Total HSP27 DuoSet IC ELISA (Catalog No. DYC1580, R&D Systems, Minneapolis, MN, USA).

Techniques: Clinical Proteomics

Journal: Annals of Laboratory Medicine

Article Title: In vitro Stability of Heat Shock Protein 27 in Serum and Plasma Under Different Pre-analytical Conditions: Implications for Large-Scale Clinical Studies

doi: 10.3343/alm.2016.36.4.353

Figure Lengend Snippet: Stability results for HSP27: effects of repeated freeze-thaw cycles with storage of serum/plasma samples at -80℃

Article Snippet: Circulating HSP27 concentrations were measured by using the Human Total HSP27 DuoSet IC ELISA (Catalog No. DYC1580, R&D Systems, Minneapolis, MN, USA).

Techniques: Clinical Proteomics

Journal: Annals of Laboratory Medicine

Article Title: In vitro Stability of Heat Shock Protein 27 in Serum and Plasma Under Different Pre-analytical Conditions: Implications for Large-Scale Clinical Studies

doi: 10.3343/alm.2016.36.4.353

Figure Lengend Snippet: In vitro stability of HSP27 under different pre-analytical conditions: (A) relative analyte stability in serum samples and (B) plasma samples stored for 2-6 hr at 4℃ with immediate and delayed sample processing; and (C) the effect of repeated freeze-thaw cycles on HSP27 serum and plasma concentrations. Graphs show relative analyte recoveries at distinct time points (each dot represents the mean analyte concentrations relative to the baseline values of 10 healthy individuals; whiskers indicate standard deviation).

Article Snippet: Circulating HSP27 concentrations were measured by using the Human Total HSP27 DuoSet IC ELISA (Catalog No. DYC1580, R&D Systems, Minneapolis, MN, USA).

Techniques: In Vitro, Clinical Proteomics, Standard Deviation

Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Activation of the Heat Shock Response in Familial Amyloidotic Polyneuropathy

doi: 10.1097/nen.0b013e31816fd648

Figure Lengend Snippet: FIGURE 1. Transthyretin (TTR) deposition induces heat shock transcription factor 1 (HSF 1) activation and increases heat shock proteins (Hsp) expression in situ. (A) Human salivary glands from normal control individuals (n = 3) and from human familial amyloidotic polyneuropathy (FAP) patients (n = 5) were immunostained for HSF 1. Greater expression and translocation to the nucleus (arrow) were observed in FAP patient samples. Scale bar = 50 Km (upper panels). Salivary glands from 6-month-old transgenic mice without TTR deposition (TTRj; n = 4) and with TTR deposition (TTR+; n = 5) were analyzed for HSF 1 expression. Only mice with TTR deposits had HSF 1 upregulation and translocation to the nucleus (arrow). Scale bar = 100 Km (bottom panels). (B) Greater expression of Hsp70 in skin from FAP patients (n = 7) compared with controls (n = 3; upper panels). Greater Hsp70 expression in SG samples from TTR positive transgenic (TTR+; n = 7) compared with TTR negative transgenic (TTRj; n = 7) mice. Scale bar = 50 Km (bottom panels). Graphs demonstrate semiquantitative analysis of Hsp70 expression shown as the average of percentage of occupied area with substrate color T standard deviation. *, p G 0.001. (C) Heat shock protein 27 expression in the peripheral nerve is upregulated in advanced stages of FAP. Normal control (n = 5) and FAP 0 (n = 5) have similarly low Hsp27 expression, whereas FAP 1 (n = 6), FAP 2 (n = 4), and FAP 3 (n = 2) have significantly greater expression. Scale bar = 30 Km. The graph demonstrates semiquantitative analysis of Hsp27 expression shown as the percentage of occupied area with substrate color T standard deviation. *, p G 0.02, †, p G 0.01. (D) Increased Hsp in FAP tissues is associated with extracellular TTR deposition. Human FAP skin samples show extracellular TTR deposition, whereas Hsp70 is detected inside gland epithelial cells. Normal human skin does not show TTR, and Hsp70 is undetectable. Arrows with small closed arrowheads point to TTR, and arrows with larger open arrowheads indicate Hsp70 (upper panels). Human FAP peripheral nerve has extracellular TTR deposits (smaller, closed arrowheads; green); Hsp27 expression is in the axons (larger, open arrowheads; red; bottom panels). Scale bar = 20 Km.

Article Snippet: Incubation with hsp70 antibody (Stressgen), diluted 1:200, or with hsp27 (Santa Cruz Biotechnology), diluted 1:100, was performed, followed by incubation with the corresponding secondary Alexa Fluor antibodies (Molecular Probes).

Techniques: Activation Assay, Expressing, In Situ, Control, Translocation Assay, Transgenic Assay, Standard Deviation

Journal: Cellular and Molecular Life Sciences

Article Title: The diterpenoid alkaloid noroxoaconitine is a Mapkap kinase 5 (MK5/PRAK) inhibitor

doi: 10.1007/s00018-010-0452-1

Figure Lengend Snippet: Compound 11 inhibits Hsp27 phosphorylation by MK3 and MK5, but not by MK2. Recombinant Hsp27 was incubated with purified MK2, MK3, or MK5 in the presence or absence of compound 11 . Hsp27 phosphorylation at serine residue 78 (p-Ser78 Hsp27) was monitored by western blot with phosphoserine-78 specific antibodies. Lanes 1 , 3 , and 5 : Hsp27 incubated with purified MK5, MK3, and MK2, respectively. Lanes 2 , 4 , and 6 : as lanes 1 , 3 , and 5 but in the presence of 20 μM noroxoaconitine (compound 11 )

Article Snippet: Recombinant Hsp27 was purchased from Signal Chem (Richmond, BC, Canada).

Techniques: Phospho-proteomics, Recombinant, Incubation, Purification, Residue, Western Blot

Journal: Journal of orofacial orthopedics = Fortschritte der Kieferorthopadie : Organ/official journal Deutsche Gesellschaft fur Kieferorthopadie

Article Title: Impact of phosphorylation of heat shock protein 27 on the expression profile of periodontal ligament fibroblasts during mechanical strain.

doi: 10.1007/s00056-022-00391-w

Figure Lengend Snippet: Fig. 3 Heat shock protein 27 (HSP27) phosphorylation was upregulated after compression and transfection of pHSP27+ (a). Transfection of pHSP27+ reduced the cell number (b) and increased lactate dehydrogenase (LDH) release (c) in periodontal ligament fibroblasts (PDLF) without and with compression. Statistics: Welch analysis of variance (ANOVA) with Dunnett’s T3 multiple comparisons tests; *P< 0.05; **P< 0.01; ***P< 0.001 Abb. 3 Die HSP27(Hitzeschockprotein 27)-Phosphorylierung wurde nach Kompression und Transfektion von pHSP27+ hochreguliert (a). Die Transfektion von pHSP27+ reduzierte die Zellzahl (b) und erhöhte die LDH(Laktatdehydrogenase)-Freisetzung (c) in PDLF (parodontale Li- gamentfibroblasten) ohne und mit Kompression. Statistik: Welch-ANOVA („analysis of variance“) mit Dunnett-T3 Post-hoc-Test; *p< 0,05; **p< 0,01; ***p< 0,001

Article Snippet: This was followed by incubation with the diluted primary antibodies (HSP27 1:3000 (AF1580, R&D Systems, Minneapolis, MN, USA); pHSP27 1:500 (AF2314, R&D Systems, Minneapolis, MN, USA), PTGS2 1:2500 (PA5-1817, Thermo Fisher Scientific, Waltham, MA, USA), RANKL 1:2000 (TA306362, OriGene, Rockville, MD, USA); ACTIN 1:1000 (E1C602, ABIN274248, Antibody-online, Aachen, Germany)).

Techniques: Phospho-proteomics, Transfection

Journal: The Journal of Cell Biology

Article Title: Endorepellin causes endothelial cell disassembly of actin cytoskeleton and focal adhesions through α2β1 integrin

doi: 10.1083/jcb.200401150

Figure Lengend Snippet: Schematic model representing the regulation of actin stress fibers and focal adhesions by ER. The model shows activation of a signaling cascade that involves ER interaction with α2β1, activation of cAMP, PKA, and FAK, followed by the transient activation of p38 MAPK and Hsp27, and ultimately disassembly of actin stress fibers and focal adhesions. The various blocking agents are also illustrated. For additional details see the text.

Article Snippet: Antibodies against Erk1/2 (Promega), P-Erk1/2 (Cell Signaling), FAK (BD Biosciences), P-FAK(Y397) (Upstate Biotechnology), p38 and P-p38 (Cell Signaling), total Hsp27 and P-Hsp27 (Santa Cruz Biotechnology, Inc.) were also used in immunoblottings.

Techniques: Activation Assay, Blocking Assay

Journal: Biomedicines

Article Title: Heat Shock Protein 27 Affects Myeloid Cell Activation and Interaction with Prostate Cancer Cells

doi: 10.3390/biomedicines10092192

Figure Lengend Snippet: Oxidative stress-induced Hsp27 and its effects in individual cell lines. ( a ) fold-change increase in oxidative stress-induced Hsp27 release after 48 h, 72 h, and 96 h in LNCaP and PC-3 cells; ( b ) representative photograph of the resazurin-based metabolic activity assay; ( c , d ) normalized metabolic activity of prostate cancer cells 96 h ( c ) and myeloid cells directly (4 h) and 24 h ( d ) following exposure to Hsp27; ( e ) representative flow cytometry dot plot of Hsp27-treated HL-60 cells; ( f ) normalized number of viable myeloid cells determined via flow cytometry directly and 24 h after Hsp27 exposure; ( g ) representative flow cytometry overlay histograms of mitotracker orange fluorescence in HL-60 cells; ( h ) normalized mean fluorescence intensity of dichlorofluorescein (DCF), aminophenyl fluorescein (APF), and mitotracker orange (MTO) in myeloid cells determined by flow cytometry; ( i ) normalized chemokine, cytokine, and growth-factor secretion profile 96 h after Hsp27 exposure in myeloid cells (additional soluble mediators investigated were below the limit of detection, including IL-1β, TNF-α, IFN-γ, IL-12p70, and IL-10). Data are representative of at least three experiments; statistical analysis was performed using one-way ( c ) or two-way ANOVA ( i ) with p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

Article Snippet: Heat shock protein 27 (Hsp27) concentration was measured using the Human Hsp27 DuoSet ELISA kit (Cat# DY1580; RnD Systems, Wiesbaden, Germany) according to the manufacturer’s protocol.

Techniques: Metabolic Assay, Activity Assay, Flow Cytometry, Fluorescence

Journal: Biomedicines

Article Title: Heat Shock Protein 27 Affects Myeloid Cell Activation and Interaction with Prostate Cancer Cells

doi: 10.3390/biomedicines10092192

Figure Lengend Snippet: Co-culture metabolic activity and release profiles. ( a ) study protocol of the co-culture of prostate cancer cell lines with myeloid cell lines treated with Hsp27; ( b ) normalized metabolic activity of Hsp27 treated co-cultures; ( c ) normalized chemokine, cytokine, and growth-factor secretion profile 96 h after Hsp27 exposure in co-cultures. Data are representative of at least three experiments; statistical analysis was performed using one-way ANOVA ( b ) with p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

Article Snippet: Heat shock protein 27 (Hsp27) concentration was measured using the Human Hsp27 DuoSet ELISA kit (Cat# DY1580; RnD Systems, Wiesbaden, Germany) according to the manufacturer’s protocol.

Techniques: Co-Culture Assay, Activity Assay

Journal: Biomedicines

Article Title: Heat Shock Protein 27 Affects Myeloid Cell Activation and Interaction with Prostate Cancer Cells

doi: 10.3390/biomedicines10092192

Figure Lengend Snippet: Hsp27-modified surface marker expression on myeloid–prostate cancer cell co-cultures. ( a ) representative flow cytometry overlay histograms of CD163 fluorescence in THP-1 cells co-cultured with PC-3 cells; ( b – h ) normalized mean fluorescence intensities of THP-1 cells obtained from monoculture (mc) or prostate cancer co-cultures (cc) 96 h after Hsp27 exposure, determined using multicolor flow cytometry; ( i ) representative flow cytometry overlay histograms of CD71 fluorescence in HL-60 cells co-cultured with LNCaP cells; ( j – o ) normalized mean fluorescence intensities of HL-60 cells obtained from monoculture (mc) or prostate cancer co-cultures (cc) 96 h after Hsp27 exposure, determined using multicolor flow cytometry. Data are representative of at least three experiments; statistical analysis was performed using one-way ANOVA with p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

Article Snippet: Heat shock protein 27 (Hsp27) concentration was measured using the Human Hsp27 DuoSet ELISA kit (Cat# DY1580; RnD Systems, Wiesbaden, Germany) according to the manufacturer’s protocol.

Techniques: Modification, Marker, Expressing, Flow Cytometry, Fluorescence, Cell Culture

Journal: Biomedicines

Article Title: Heat Shock Protein 27 Affects Myeloid Cell Activation and Interaction with Prostate Cancer Cells

doi: 10.3390/biomedicines10092192

Figure Lengend Snippet: High-content imaging of THP-1 cells co-cultured with prostate cancer cells under Hsp27 treatment. ( a ) representative overlay images of untreated (control) and Hsp27-treated co-cultures (cc) of CellTrace Violet-labeled prostate cancer cells (blue) and CellTrace Far Red-labeled THP-1 cells (red), with SYTOX Green (green) as dead cell marker at 96 h; ( b ) PCAs of several morphology properties quantified by algorithm-based image analysis of THP-1 cells; ( c – l ) algorithm-based image quantification of THP-1 total cell count ( c , d ), the total aggregate area in µm 2 ( e , f ), number of cells interacting with prostate cancer cells ( g , h ), distance to the nearest prostate cancer cell in µm ( i , j ), and mean fluorescence intensity (MFI) of dead prostate cancer cell material internalized by THP-1 cells ( k , l ). Data are presented as mean + SE. Statistical analysis was performed using unpaired, two-tailed t-test with p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). ns = not significant. Scale bar is 100 µm.

Article Snippet: Heat shock protein 27 (Hsp27) concentration was measured using the Human Hsp27 DuoSet ELISA kit (Cat# DY1580; RnD Systems, Wiesbaden, Germany) according to the manufacturer’s protocol.

Techniques: Imaging, Cell Culture, Control, Labeling, Marker, Cell Counting, Fluorescence, Two Tailed Test

Journal: Biomedicines

Article Title: Heat Shock Protein 27 Affects Myeloid Cell Activation and Interaction with Prostate Cancer Cells

doi: 10.3390/biomedicines10092192

Figure Lengend Snippet: High-content imaging of HL-60 cells co-cultured with prostate cancer cells under Hsp27 treatment. ( a ) representative overlay images of untreated (control) and Hsp27-treated co-cultures (cc) of CellTrace Violet-labeled prostate cancer cells (blue) and CellTrace Far Red-labeled HL-60 cells (red), with SYTOX Green (green) as dead cell marker at 96 h; ( b ) PCAs of several morphology properties quantified by algorithm-based image analysis of HL-60 cells; ( c – l ) algorithm-based image quantification of HL-60 total cell count ( c , d ), the total aggregate area in µm 2 ( e , f ), number of cells interacting with the prostate cancer cells ( g , h ), distance to the nearest prostate cancer cell in µm ( i , j ), and mean fluorescence intensity (MFI) of dead prostate cancer cell material internalized by HL-60 cells ( k , l ). Data are presented as mean + SE. Statistical analysis was performed using unpaired, two-tailed t -test with p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). ns = not significant. Scale bar is 100 µm.

Article Snippet: Heat shock protein 27 (Hsp27) concentration was measured using the Human Hsp27 DuoSet ELISA kit (Cat# DY1580; RnD Systems, Wiesbaden, Germany) according to the manufacturer’s protocol.

Techniques: Imaging, Cell Culture, Control, Labeling, Marker, Cell Counting, Fluorescence, Two Tailed Test

Journal: PloS one

Article Title: Human-derived physiological heat shock protein 27 complex protects brain after focal cerebral ischemia in mice.

doi: 10.1371/journal.pone.0066001

Figure Lengend Snippet: Figure 1. Characterization of hHSP27. A–B, Isolated human heat shock protein 27 (hHSP27) or recombinant HSP27 (rHSP27) proteins (1 mg or 10 mg) were separated by SDS-PAGE (A) and native-PAGE (B) and stained with Coomassie brilliant blue. C, hHSP27 proteins were immunoblotted with antibodies against HSP27, phosphorylated S15 HSP27, S78 HSP27, and S82 HSP27. D, hHSP27 proteins were separated by native-PAGE and immunoblotted with antibodies against ab-crystallin and HSP20. E, rHSP27 (10 ng), hHSP27 (10 ng), ab-crystallin (5 ng), and HSP20 (5 ng) were separated by SDS-PAGE followed by immunoblotting with antibodies against HSP27, ab-crystallin, and HSP20. doi:10.1371/journal.pone.0066001.g001

Article Snippet: The observers of the behavioral tests were blinded to the treatment groups, and mice of the various groups were randomized during a given testing period. hHSP27 and HSP27 Antibody, HSP27 Elution Peptide Administration or Recombinant HSP27 Administration Mice received intravenous injections of 50 mg of hHSP27 mixed with 50 or 500 mg of HSP27-N1 or -C1 antibody 1 h after reperfusion (n= 3 in each group), 5 or 50 mg of HSP27-N1 and - C1 peptides, which were used in the elution, intravenously 1 h after reperfusion (n= 3 in each group), or 50 mg of recombinant HSP27 (rHSP27; Acris Antibodies GmbH) 1 h after reperfusion (n= 3).

Techniques: Isolation, Recombinant, SDS Page, Clear Native PAGE, Staining, Western Blot

Journal: PloS one

Article Title: Human-derived physiological heat shock protein 27 complex protects brain after focal cerebral ischemia in mice.

doi: 10.1371/journal.pone.0066001

Figure Lengend Snippet: Figure 4. Anti-HSP27 antibody and dephosphorylation inhibit hHSP27 neuroprotective effects. A, Infarct volumes in control, hHSP27 (50 mg), hHSP27 plus HSP27 antibody cocktails, HSP27 elution peptide, recombinant HSP27, and dephosphorylated hHSP27 groups. Data are means6SEM of 3 mice in each group. **P,0.001 vs. controls. B– D, Dephosphorylated and phosphorylated hHSP27 proteins were separated by SDS- PAGE (B) and native-PAGE (C), stained with Coomassie brilliant blue (B,C), and immunoblotted with anti-phosphorylated S15 HSP27, S78 HSP27, and S82 HSP27 antibodies (D). E, Photomicrographs of infarct areas stained with cresyl violet in hHSP27 and dephosphorylated hHSP27 groups prepared 24 h after reperfusion. Scale bar = 1 mm. hHSP27, human heat shock protein. doi:10.1371/journal.pone.0066001.g004

Article Snippet: The observers of the behavioral tests were blinded to the treatment groups, and mice of the various groups were randomized during a given testing period. hHSP27 and HSP27 Antibody, HSP27 Elution Peptide Administration or Recombinant HSP27 Administration Mice received intravenous injections of 50 mg of hHSP27 mixed with 50 or 500 mg of HSP27-N1 or -C1 antibody 1 h after reperfusion (n= 3 in each group), 5 or 50 mg of HSP27-N1 and - C1 peptides, which were used in the elution, intravenously 1 h after reperfusion (n= 3 in each group), or 50 mg of recombinant HSP27 (rHSP27; Acris Antibodies GmbH) 1 h after reperfusion (n= 3).

Techniques: De-Phosphorylation Assay, Control, Recombinant, SDS Page, Clear Native PAGE, Staining