All chemicals were purchased from either Sigma‐Aldrich (Poole, UK) or Fisher Scientific UK Ltd (Loughborough, UK), except where statedotherwise. Antibodies against phospho‐Akt (Ser473), phospho‐Akt (Thr308), Akt, phospho‐4EBP‐1 (Thr37/46), 4EBP‐1, phospho‐eIF2α (Ser51), eIF2α, phospho‐p38 MAPK (Thr180/Tyr182), p38 kinase, phospho‐AMP‐activating kinase(AMPK)α(Thr172), AMPKα, phospho‐heat shock protein (HSP)27 (Ser82) and HSP27 were obtained from Cell Signalling Technology (NEB, Hitchin, UK). Antibodies against XBP‐1, GRP94 and HSP60 were obtained from Abcam (Cambridge, UK), and HSP90 and HSP70 were obtained from Enzo Life Science (Exeter, UK). Anti‐GRP78 was from Transduction Laboratories (BD Biosciences, Oxford, UK) and anti‐β‐actin was obtained from Sigma‐Aldrich. OxPhos Complex Kit for mitochondrial electron transport chain (ETC) subunits was obtained from Invitrogen (Renfrew, UK).
Hsp27
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, China
HSP27 is a lab equipment product offered by Cell Signaling Technology. It is a heat shock protein that functions as a molecular chaperone, assisting in the folding and stabilization of other proteins.
Automatically generated - may contain errors
Lab products found in correlation
P hsp27, by Cell Signaling Technology (7 mentions)
Hsp70, by Cell Signaling Technology (5 mentions)
Gapdh, by Cell Signaling Technology (5 mentions)
P38 mapk, by Cell Signaling Technology (4 mentions)
Bcl 2, by Cell Signaling Technology (4 mentions)
Cleaved caspase 3, by Cell Signaling Technology (4 mentions)
Phospho hsp27, by Cell Signaling Technology (3 mentions)
Hsp60, by Cell Signaling Technology (3 mentions)
Hsp40, by Cell Signaling Technology (3 mentions)
Sb203580, by Merck Group (3 mentions)
Phospho p38 mapk thr180 tyr182, by Cell Signaling Technology (2 mentions)
P mk2, by Cell Signaling Technology (2 mentions)
Verso cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
C1000 thermal cycler, by Bio-Rad (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Phospho p38, by Cell Signaling Technology (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Vimentin, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Tnf α, by R&D Systems (2 mentions)
39 protocols using hsp27
1
Quantifying Cellular Signaling Pathways
2
Western Blot Analysis of Cell Signaling Pathways
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Total protein
extracts were generated using lysis buffer (50 mM Tris-HCl, pH 7.4,
150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1
mM EDTA) supplemented with PhosSTOP and Complete Phosphatase/Protease
Inhibitor Cocktails (Roche Diagnostics GmbH, Mannheim, Germany). Protein
extracts (20–25 μg per sample) were loaded onto sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels
and transferred electrophoretically to poly(vinylidene fluoride) (PVDF)
membranes. The following primary antibodies were used at a dilution
of 1:1000: ERK1/2 (p44/42) (ab17942, Abcam), p-ERK1/2 (p-p44/42) (Thr202/Tyr204)
(9101, Cell Signaling), MNK1 (2195, Cell Signaling), p-MNK1 (Thr197/202)
(2111, Cell Signaling), eIF4E (9742, Cell Signaling), p-eIF4E (p-Ser
209) (9741, Cell Signaling or NBP2-66802, Novus Biologicals), p38alpha
(9218, Cell Signaling), p38beta (2339, Cell Signaling), p-p38 (4511,
Cell Signaling), p-ATF2 (27934, Cell Signaling), Hsp27 (2402, Cell
Signaling), p-Hsp27 (S82) (2401, Cell Signaling), and p-p90 RSK (Thr573)
(9346, Cell Signaling). The primary HRP-conjugated antibody anti-β-actin
(Calbiochem) was used at a dilution of 1:20 000. Anti-mouse
and anti-rabbit HRP secondary antibodies were from Pierce and used
at a dilution of 1:10 000. Immunodetection of proteins was
performed using ECL Western Blotting Detection Reagents (GE Healthcare,
Buckinghamshire, U.K.).
extracts were generated using lysis buffer (50 mM Tris-HCl, pH 7.4,
150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1
mM EDTA) supplemented with PhosSTOP and Complete Phosphatase/Protease
Inhibitor Cocktails (Roche Diagnostics GmbH, Mannheim, Germany). Protein
extracts (20–25 μg per sample) were loaded onto sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels
and transferred electrophoretically to poly(vinylidene fluoride) (PVDF)
membranes. The following primary antibodies were used at a dilution
of 1:1000: ERK1/2 (p44/42) (ab17942, Abcam), p-ERK1/2 (p-p44/42) (Thr202/Tyr204)
(9101, Cell Signaling), MNK1 (2195, Cell Signaling), p-MNK1 (Thr197/202)
(2111, Cell Signaling), eIF4E (9742, Cell Signaling), p-eIF4E (p-Ser
209) (9741, Cell Signaling or NBP2-66802, Novus Biologicals), p38alpha
(9218, Cell Signaling), p38beta (2339, Cell Signaling), p-p38 (4511,
Cell Signaling), p-ATF2 (27934, Cell Signaling), Hsp27 (2402, Cell
Signaling), p-Hsp27 (S82) (2401, Cell Signaling), and p-p90 RSK (Thr573)
(9346, Cell Signaling). The primary HRP-conjugated antibody anti-β-actin
(Calbiochem) was used at a dilution of 1:20 000. Anti-mouse
and anti-rabbit HRP secondary antibodies were from Pierce and used
at a dilution of 1:10 000. Immunodetection of proteins was
performed using ECL Western Blotting Detection Reagents (GE Healthcare,
Buckinghamshire, U.K.).
3
Gene and Protein Expression Analysis
4
Platelet Activation Signaling Pathways
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Collagen, thrombin, ADP and luciferin/luciferase were provided by the Chrono-Log Corporation. FITC-phalloidin, H2O2 and mepacrine were purchased from Sigma-Aldrich; Merck KGaA. Antibodies against phospho-ERK1/2 (catalog no. 4370S), phospho-p38 (catalog no. 4511S), phospho-HSP27 (catalog no. 2401S), total-p38 (catalog no. 8690S), total-ERK (catalog no. 4695S) and HSP27 (catalog no. 95357S) were purchased from Cell Signaling Technology Inc. β-actin monoclonal antibody (catalog no. 66009-1-Ig) was obtained from ProteinTech Group, Inc. The antibody for integrin β3 (D-11) (catalog no. sc-365679) was obtained from Santa Cruz Biotechnology Inc. PAC-1 antibodies (catalog no. MA5-28564) were from Invitrogen; Thermo Fisher Scientific, Inc., and CD62P antibodies (catalog no. 555524) were from BD Biosciences.
5
Cisplatin-Induced HSP Expression in MSCs
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MSCs were treated with 1000 ng/mL cisplatin for 4 hours, and cells were harvested 12 and 24 hours later. Each sample containing 10 μg of total protein from whole-cell lysates was run on a polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Millipore, Darmstadt, Germany). Membranes were probed with primary antibodies against HSP90α (Abcam, Cambridge, UK), HSP90β (Abcam), HSP72 (LifeSpan Biosciences, Eching, Germany), HSP27 (Cell Signaling Technology, Leiden, Netherlands), HSP60 (Cell Signaling Technology), and HSP10 (Santa Cruz, Heidelberg, Germany). β-actin was used as a loading control. Blots were visualized on X-ray film using a horseradish-peroxidase kit (Cell Signaling Technology).
6
Investigating Epithelial-Mesenchymal Transition
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RPMI-1640 and fetal bovine serum (FBS) was acquired from Gibco-BRL (Gaithersburg, MD, USA). 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Recombinant TGF-β1 and TNF-α were obtained from R&D systems (Minneapolis, MN, USA). Matrigel was purchased from BD Biosciences (San Jose, CA, USA). Antibodies against E-cadherin, N-cadherin, Vimentin, HSP27, Snail, NF-κB p65, IκBα, phosphorylation of IκBα, Histone H3 and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Other chemicals used were of analytical grade from commercial sources.
7
Multiplex Protein Profiling of Cellular Pathways
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Analysis was performed by DigiWest multiplex protein profiling, as described previously [12 (link)]. The following primary antibodies were used: pH 3-specific antibody: protein kinase B phosphorylation (pAKT-specific antibody: 12,178 (D5G4), Cell Signaling Technology Cambridge, UK, 1:200), protein kinase B (AKT-specific antibody: 9272S, Cell Signaling Technology, 1:200), heat shock protein 27 (HSP27-specific antibody: 2402 (G31), Cell Signaling Technology, 1:200), Survivin (Survivin-specific antibody: 2808S (71G4B7), Cell Signaling Technology, 1:200), Signal transducer and activator of transcription 3 (STAT3-specific antibody: 9139S (124H6), Cell Signaling Technology, 1:200), Cyclin-dependent kinase 4 (CDK4-specific antibody: 12790S (D9G3E) (1272), Cyclin D1 (Cyclin D1-specific antibody: ab134175 (EPR2241), Abcam, Cambridge, UK, 1:200), cyclin-dependent kinase inhibitor 1 (p21-specific antibody: ab109520 (EPR362) Abcam, 1:200), p-Histone H3 (pH3-specific antibody: 9701, Cell Signaling Technology, 1:200), p53 phosphorylation (p-p53-specific antibody: 9284, Cell Signaling Technology, 1:200), Retinoblastoma protein (Rb-specific antibody: ab181616, Abcam, 1:200). Values of PAM-treated cells were normalized to the control group.
8
Western Blot Immunodetection of Molecular Chaperones
9
Western Blot Analysis of Cell Signaling
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Tissue homogenization and SDS–PAGE electrophoresis and immunoblotting were carried out as previously described (Tjoa et al., 2006 (link)). Primary antibodies against phospho-p38, phospho-AKT, TNF-α, Hsp27 and IκB were from Cell Signaling (all used at 1:1000; New England Biolabs, Hitchin, UK); anti-HIF-1α was from Novus Biologicals (used at 1:500; Cambridge, UK). Proteins were revealed and quantified using Image J software (National Institutes of Health, http://rsb.info.nih.gov/ij/ ). Protein loading was normalized against β-actin or Poncaeu S staining. Values are expressed as a percentage of the control lysate (100%) for each experiment. Western blot measurements were analysed using ANOVA, and the Protected Least Significant Differences post hoc test (Fisher's post hoc test). Differences between two groups were evaluated using a Fisher's post hoc test. Results were considered significant at P < 0.05.
10
Immunoblotting Analysis of Stress Proteins
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This assay was performed as previously described. Briefly, treated cells were washed twice with PBS and then lysed in ice-cold cell lysis buffer (Cell Signaling Technology, Beverly, MA) containing 1 mM PMSF. After 1 h of incubation, samples were centrifuged at 12,000 g for 15 min. Supernatants were then collected. Proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes, which were incubated with appropriate antibodies and visualized using an enhanced chemiluminescence system (GE Healthcare, Buckinghamshire, UK). Antibodies used in the current study were HO-1, Nrf2, COX-2, HSP-27, HSP-60, HSP-70, all purchased from Cell Signaling Technology.
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