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: Biochimica et biophysica acta

Article Title: Cx43 increases serum induced filopodia formation via activation of p21-activated protein kinase 1.

doi: 10.1016/j.bbamcr.2015.08.004

Figure Lengend Snippet: Fig. 7. Cx43 expression enhanced phosphorylation of Hsp27 and F-actin polymerization. (A) Cx43 expression augmented Hsp27 phosphorylation in response to serum stimulation. This effect was mediated by p38, since the p38 inhibitor SB203580 (+SB) reduced the Hsp27 phosphorylation. (B) Inhibitory effect of the p38 inhibitor (+SB) on the amount of phosphorylated Hsp27 15 min after serum incubation (D: DMSO treated, SB: treated with p38 inhibitor). (C) The F-actin/G-actin ratio was significantly increased in Cx43 ex- pressing HeLa cells after stimulation with 10% NBCS for 3 h (mean ± SEM; n = 5 indepen- dent experiments; p b 0.05).

Article Snippet: Primary antibodies used: anti-Cx43 (SigmaAldrich, 1:3000), anti-GFP (1:500; Abcam), anti-phospho Hsp27 S82 (Cell Signaling, 1:1000), antiphospho αPAK Thr423 (1:500; Santa Cruz), anti-phospho p38 (T180/ Y182) and anti-GAPDH (Chemicon, 1:20,000)were diluted in 5% bovine serum albumin (BSA, AppliChem) in PBS-0.1% Tween.

Techniques: Expressing, Phospho-proteomics, Incubation

Journal: Biochimica et biophysica acta

Article Title: Cx43 increases serum induced filopodia formation via activation of p21-activated protein kinase 1.

doi: 10.1016/j.bbamcr.2015.08.004

Figure Lengend Snippet: Fig. 8. Proposed model of Cx43 mediated augmentation of filopodia formation and cell migration. Growth factor stimulation leads to an activation of PAK1 via activation of small GTPases Rac1 and Cdc42. The interaction with Cx43 enables the enzyme to be more activated and allows therefore also a stronger phosphorylation of the subsequent downstream molecules p38 and Hsp27. Due to the enhanced phosphorylation, the actin capping function of Hsp27 is abolished and phosphorylated Hsp27 favors thereby the actin polymerization and promotes the cell migration.

Article Snippet: Primary antibodies used: anti-Cx43 (SigmaAldrich, 1:3000), anti-GFP (1:500; Abcam), anti-phospho Hsp27 S82 (Cell Signaling, 1:1000), antiphospho αPAK Thr423 (1:500; Santa Cruz), anti-phospho p38 (T180/ Y182) and anti-GAPDH (Chemicon, 1:20,000)were diluted in 5% bovine serum albumin (BSA, AppliChem) in PBS-0.1% Tween.

Techniques: Migration, Activation Assay, Phospho-proteomics

Journal: Journal of biomechanics

Article Title: Mechanical strain induced phospho-proteomic signaling in uterine smooth muscle cells

doi: 10.1016/j.jbiomech.2018.03.040

Figure Lengend Snippet: This tonic mechanical strain revealed phosphorylation of ERK1/2 and HSPB1. Additionally, we saw increased phosphorylation of p38 kinase, an activator of HSPB1 and a protein predicted by IPA to be activated. Statistical analysis of all blots was performed by normalization of phosphorylation to total protein with (*) representing a p value of <0.001.

Article Snippet: Samples were electrophoresed on sodium dodecyl sulfate 4% to 20% gradient polyacrylamide gels and western blotted according to manufacturer instructions (BioRad, Hercules, Ca) with the following primary antibodies: p38 MAPK (D13E1) XP ® #8690; Phospho-p38 (Thr180/Tyr182) (D3F9) XP ® #4511; HSP27 Antibody Sampler Kit #12594 were from Cell Signaling Technology (Beverly, MA); Primary antibodies were detected with IRDye 680LT goat anti-rabbit (LiCor Biosciences, Lincoln, NE) or IRDye 800LT donkey anti-mouse (827-08364, Rockland Immunochemicals, Gilbertsville, PA).

Techniques:

Journal: Journal of biomechanics

Article Title: Mechanical strain induced phospho-proteomic signaling in uterine smooth muscle cells

doi: 10.1016/j.jbiomech.2018.03.040

Figure Lengend Snippet: Experimentally determined phosphorylated proteins at 5 min (green with orange P) with pathway proteins (blue) that were not experimentally observed to be phosphorylated after 5 min of mechanical strain. Associations based on literature searches and phospho-site annotation (HSPB1 represented here as HSP27).

Article Snippet: Samples were electrophoresed on sodium dodecyl sulfate 4% to 20% gradient polyacrylamide gels and western blotted according to manufacturer instructions (BioRad, Hercules, Ca) with the following primary antibodies: p38 MAPK (D13E1) XP ® #8690; Phospho-p38 (Thr180/Tyr182) (D3F9) XP ® #4511; HSP27 Antibody Sampler Kit #12594 were from Cell Signaling Technology (Beverly, MA); Primary antibodies were detected with IRDye 680LT goat anti-rabbit (LiCor Biosciences, Lincoln, NE) or IRDye 800LT donkey anti-mouse (827-08364, Rockland Immunochemicals, Gilbertsville, PA).

Techniques:

Journal: Cellular microbiology

Article Title: Coiled-coil domains enhance the membrane association of Salmonella type III effectors.

doi: 10.1111/j.1462-5822.2011.01635.x

Figure Lengend Snippet: Fig. 6. The CC domain directs efficient membrane association of SopB. A. HeLa cells were transfected with plasmids expressing EGFP-HuSopB, EGFP-HuSopB C460S, EGFP-HuSopBDCC or EGFP-HuSopB-L3D for 20 h. Monolayers were fixed and DNA stained with DRAQ5. Confocal microscope images show EGFP-SopB fusions in grey scale and DNA in blue. Scale bar, 10 mm. B. HeLa cells were transfected with plasmids expressing Myc-SopB, Myc-SopB C460S, Myc-SopBDCC or Myc-SopB-L3D, harvested and subject to sequential detergent fractionation. Equal volumes of the saponin-soluble (S, cytosolic proteins) and TX-100-soluble (T, membrane proteins) fractions were separated by SDS-PAGE and subject to immunoblotting with antibodies against SopB, LAMP-1 (membranes) and Hsp27 (cytosol). C. HeLa cells were infected with DsopB-sigE bacteria harbouring pWSKDE-GSK (SopB), pWSKDEDCC-GSK (DCC) or pWSKDE-L3D-GSK (L3D). At 1 h p.i., infected cells were mechanically fractionated and subject to low-speed centrifugation to remove intact bacteria, and then high-speed ultracentrifugation to separate host cell membranes (M) from cytosol (C). Equal volumes of each fraction were subject to immunoblotting with antibodies against phospho-Ser9-GSK3b (translocated SopB-GSK), LAMP-1 (membranes) and Hsp27 (cytosol).

Article Snippet: Hildreth was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa; 1:5000) and mouse monoclonal anti-Hsp27 antibodies (G31, Cell Signaling Technology, 1:20 000) as described previously (Knodler et al., 2009).

Techniques: Membrane, Transfection, Expressing, Staining, Microscopy, Fractionation, SDS Page, Western Blot, Infection, Bacteria, Centrifugation

Journal: Cellular microbiology

Article Title: Coiled-coil domains enhance the membrane association of Salmonella type III effectors.

doi: 10.1111/j.1462-5822.2011.01635.x

Figure Lengend Snippet: Fig. 7. CC domains mediate the membrane association of other Salmonella effectors. A. HeLa cells were transfected with plasmids expressing EGFP-PipB2, EGFP-PipB2 YLM3D, EGFP-SseJ, EGFP-SseJ NLV3D, EGFP-SopD2 or EGFP-SopD2 YYK3D for 24 h. Monolayers were fixed and DNA stained with DRAQ5. Confocal microscope images show EGFP-effector fusions in grey scale and DNA in blue. Scale bar, 10 mm. B. HeLa cells were transfected as described in (A). Cells were harvested and subject to sequential detergent fractionation. Equal volumes of the saponin-soluble (S, cytosolic proteins) and TX-100-soluble (T, membrane proteins) fractions were separated by SDS-PAGE and subject to immunoblotting with antibodies against GFP, LAMP-1 (membranes) and Hsp27 (cytosol). The asterisk indicates a non-specific band in saponin-soluble fractions that cross-reacts with the anti-GFP antibody.

Article Snippet: Hildreth was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa; 1:5000) and mouse monoclonal anti-Hsp27 antibodies (G31, Cell Signaling Technology, 1:20 000) as described previously (Knodler et al., 2009).

Techniques: Membrane, Transfection, Expressing, Staining, Microscopy, Fractionation, SDS Page, Western Blot

Journal: Cellular microbiology

Article Title: Coiled-coil domains enhance the membrane association of Salmonella type III effectors.

doi: 10.1111/j.1462-5822.2011.01635.x

Figure Lengend Snippet: Fig. 8. CC domains are not functionally interchangeable. A. HeLa cells were transfected with plasmids expressing EGFP-HuSopB or the following chimeras, EGFP-HuSopB-IpgDCC, EGFP- HuSopB-PipB2CC, EGFP-HuSopB-SseJCC or EGFP-HuSopB-SopD2CC for 24 h. Monolayers were fixed and DNA stained with DRAQ5. Confocal microscope images show EGFP-SopB fusions in grey scale and DNA in blue. Scale bar, 10 mm. B. HeLa cells were transfected as described in (A), harvested and subject to sequential detergent fractionation. Equal volumes of the saponin-soluble (S, cytosolic proteins) and TX-100-soluble (T, membrane proteins) fractions were separated by SDS-PAGE and subject to immunoblotting with antibodies against GFP, LAMP-1 (membranes) and Hsp27 (cytosol).

Article Snippet: Hildreth was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa; 1:5000) and mouse monoclonal anti-Hsp27 antibodies (G31, Cell Signaling Technology, 1:20 000) as described previously (Knodler et al., 2009).

Techniques: Transfection, Expressing, Staining, Microscopy, Fractionation, Membrane, SDS Page, Western Blot

Journal: bioRxiv

Article Title: Aging associated altered response to intracellular bacterial infections and its implication on the host

doi: 10.1101/2020.09.15.298158

Figure Lengend Snippet: A . Bacterial CFU at 16h post invasion in vehicle, NAC and AMG treated senescent HeLa cells. B . p38 MAPK inhibition decreases bacterial proliferation. Senescent HeLa cells were infected in the presence of a specific p38MAPK inhibitor, SB 202190 (SB) and bacterial CFU was determined at 16 h post invasion. C . Western blot of p38 activity by monitoring phosphorylation status of Hsp27, a downstream substrate of p38MAPK in infected and uninfected senescent cells, treated with vehicle or SB202190. D . Analysis of changes in NO levels in SB202190 treated senescent cells by Griess assay. E . Expression analysis of NOS2 in senescent cells treated with SB202190 by qRT-PCR. Values are normalized to β-actin and then wrt vehicle treated cells to obtain fold changes. F . Effect of co-inhibition of iNOS by AMG and p38 by SB202190 on intracellular bacterial proliferation. The data represents mean ± SEM from atleast three independent experiments. Statistical significance of differences was analysed by Mann-Whitney U test, * P ≤ 0.05, ** P ≤ 0.01.

Article Snippet: All the primary antibodies were from CST (Cell Signalling Technology Inc., USA) and used at 1:1000 dilution overnight at 4°C for probing protein levels viz. phospho-Hsp27 (Cat No. 9709), Hsp27 (Cat No. 95357) GAPDH (Cat No. 2118) and phospho-Chk2 (Cat no. 2197).

Techniques: Inhibition, Infection, Western Blot, Activity Assay, Griess Assay, Expressing, Quantitative RT-PCR, MANN-WHITNEY

Journal: Frontiers in Oncology

Article Title: Stromal Fibroblasts Counteract the Caveolin-1-Dependent Radiation Response of LNCaP Prostate Carcinoma Cells

doi: 10.3389/fonc.2022.802482

Figure Lengend Snippet: RT-induced stromal alterations decisively impact on adjacent prostate carcinoma (PCa) cells. PCa progression is associated with a critical shift of epithelial–stromal CAV1 expression levels. A re-expression of CAV1 within the malignant epithelial cells could be associated with epithelial-to-mesenchymal transition (EMT), an increased three-dimensional spheroid growth and RT resistance as investigated here. Concerning the related signaling, CAV1 seems to are promote adenosine monophosphate-activated protein kinase (AMPK) and potentially heat shock protein 27 (HSP27) activities, at least in combination with RT. As an assumption, increased HSP27 phosphorylations could foster (androgen-independent) androgen receptor (AR) signaling by displacing other HSPs (e.g., HSP90) from the cytosolic complex, with the AR subsequently squiring into the nucleus modulating target gene (e.g., EMT gene) expressions. It was further shown here, that PTRF (cavin-1)-deficient LNCaP PCa cells exhibit long-lasting high-density GM1 ganglioside containing signaling domains that were not affected following a gain in CAV1. In general, upon CAV1 re-expressions, either following increased cellular stress (e.g., by radiation treatment, RT) or by ectopic expression of CAV1, CAV1 can be found at the plasma membrane in non-caveolae plasma membrane domains, so-called scaffolds. Accompanied cholesterol binding reduces the amount of available cholesterol and thus the precursor for the synthesis of the steroid hormones. We therefore speculate that CAV1-dependent receptor signaling and raft-dependent endocytosis might account for signalosome-amplified signaling events that finally foster PCa cells growth, invasion and therapy resistance. Herein CAV1 Tyr14 phosphorylation seemed not to be required for the increased invasion potential of CAV1-expressing LNCaP cells, as it was shown within the present study that CAV1 Y14F-expressing LNCaP cells showed a similar EMT-phenotype but lacked increased three-dimensional spheroid growth. Similarly, non-plasma membrane-localized CAV1 was associated with an EMT phenotype while lacking increased spheroid growth. A gain of epithelial CAV1 could further be linked to increasing metabolic demands that were managed by increased glycolysis levels. Within CAV1 P132L-expressing LNCaP PCa cells, the strongest increase in glycolysis could be observed that, together with increased mitochondrial respiration rates, may compensate a reduced cholesterol utilization due to decreased cholesterol levels. RT-induced HSP27 signaling together with an increased AMPK-dependent signaling might even foster other metabolic processes to generate energy and biomolecules, e.g., fatty acid oxidation. In contrast, an activated fibroblast phenotype either following RT or potentially following ‘education’ by adjacent cancer cells will act tumor supporting, which in turn limits applied therapies. Likewise, a loss of stromal CAV1 was already shown to come along with a more reactive fibroblast phenotype as well as increased radioresistance of advanced PCa. Particularly increases in the metabolic potential as shown here following RT, and thus exocytosis of fibroblast-derived factors could not only fuel adjacent cancer cells but even comprise a feeding with resistance factors finally fostering PCa progression and therapy resistance. Thus, the loss of stromal CAV1 rather than the gain of epithelial CAV1 accounts more decisively for (radiation) therapy failure.

Article Snippet: The CAV1 (D46G3; #3267), SRC (36D10; #2109) phosphorylated SRC Tyr527 (#2105), Tyr416 (E6G4R; #59548), vimentin (D21H3; #5741), AMPK (#2532), phosphorylated AMPK Thr172 (40H9; #2535), and phosphorylated HSP27 Ser82 (D1H2F6; #9709) antibodies were from Cell Signaling Technology (Danvers, MA; all 1:1000), against TAGLN from Proteintech (Chicago, IL), and beta-actin antibody (AC-74, A2228 was from Sigma-Aldrich).

Techniques: Expressing, Clinical Proteomics, Membrane, Binding Assay, Amplification, Phospho-proteomics, Derivative Assay

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: 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