Protein extraction was performed in ice-cold RIPA buffer with a protein inhibitor cocktail. Thirty μg protein was loaded and separated on 10% SDS-PAGE gels. Protein samples were transferred onto nitrocellulose membranes (Whatman International Ltd.), stained with amido-black and photographed to verify equal loading and quality of the transfer. The membranes were subsequently incubated with blocking solution and incubated overnight at 4°C with primary antibodies against mouse p66Shc-pSer36 (Calbiochem, UK), heat shock protein 70 (Abcam, UK) or mouse actin (Sigma-Aldrich, Germany). The membranes were then incubated with appropriate horseradish-peroxidase-conjugated secondary antibodies (Dako, UK) at room temperature. Protein bands were visualised by chemiluminescence detection kit (Gibco®, Invitrogene Life Science, UK) and signals normalized to the corresponding actin optical density (Bio-Rad quantity one software, UK).
Heat shock protein 70
Manufactured by Abcam
Sourced in United Kingdom, China, Germany
Heat shock protein 70 is a molecular chaperone that assists in the folding and unfolding of proteins. It plays a crucial role in maintaining protein homeostasis within cells.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Cytiva (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Agilent Technologies (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Bicinchoninic acid protein determination kit, by Thermo Fisher Scientific (1 mentions)
C casp3, by Cell Signaling Technology (1 mentions)
N cadherin n cad, by Cell Signaling Technology (1 mentions)
C parp, by Cell Signaling Technology (1 mentions)
Vimentin vim, by Cell Signaling Technology (1 mentions)
β actin actb, by Cell Signaling Technology (1 mentions)
E cadherin e cad, by Cell Signaling Technology (1 mentions)
Mmp 2, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Gapdh, by Abcam (1 mentions)
Bcl 2, by Affinity Biosciences (1 mentions)
Hsp70, by Abcam (1 mentions)
β tubulin, by Cell Signaling Technology (1 mentions)
3 protocols using heat shock protein 70
1
Western Blot Analysis of Protein Expression
2
Protein Extraction and Western Blot Analysis from Diverse Biological Samples
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Proteins were extracted from MC38 cells, liver tissues, CRLM tissues, CRLM-adjacent tissues, and exosomes by a lysis buffer (50 mmol/L Tris, 1% NP40, 0.25% deoxycholic acid sodium salt, 150 mmol/L NaCl, 1 mmol/L EGTA), and protein concentrations were determined using a bicinchoninic acid protein determination kit (Thermo Scientific, Waltham, MA) according to the manufacturer’s instructions. Western blot analyses were performed with antibodies against SMPD3 (Santa Cruz Biotechnology), MMP2 (Cell Signaling Technology), cCASP3 (Cell Signaling Technology), cPARP (Cell Signaling Technology), BCL-2 (Affinity, Changzhou, Jiangsu, China), BAX (Cell Signaling Technology), cytochrome C (CYC) (Abcam), heat shock protein 90 (Abcam), heat shock protein 70 (Abcam), CD63 (Abcam), CD9 (Proteintech, Guangzhou, China), N-cadherin (NCAD) (Cell Signaling Technology), vimentin (VIM) (Cell Signaling Technology), E-cadherin (ECAD) (Cell Signaling Technology), CANTB (Cell Signaling Technology), CD31(Abcam), GAPDH (Abcam), Histong3 H3 (Abcam), and β-Actin (ACTB) (Cell Signaling Technology). ACTB was used as a loading control. The information on these primary antibodies is listed in Table 2 .
3
Western Blotting of Oxidative Stress Proteins
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Western blotting was performed as described recently [12] . The membranes were incubated with antibodies against 3-mercaptopyruvate sulfurtransferase (3-MST; Sigma, Taufkirchen, Germany), cystathionine-γlyase (CSE; Santa Cruz Biotechnology Inc., Heidelberg, Germany) or cystathionine-β-synthetase (CBS; Abnova, Heidelberg, Germany), hemeoxygenase-1 (HO-1; Biomol GmbH, Hamburg, Germany), heat shock protein 70 (HSP70; Abcam, Cambridge, UK), phosphorylated c-jun N-terminale kinase (pJNK), phosphorylated extracellular-signal regulated kinases (pERK1/2), phosphorylated p38 (pp38; all Cell Signaling, Frankfurt, Germany), NADPH oxidase 1 and 4 (Nox1; Nox4; Santa Cruz), or Nox2 (Becton Dickinson GmbH, Heidelberg, Germany). Normalization in order to control equal protein loading was performed by stripping and re-blotting of the membranes with glyceraldehyde-3-phosphate-dehydrogenase (GAPDH; Enzo Life Sciences GmbH, Lörrach, Germany) or βtubulin (Cell Signaling). Data represent fold induction of indicated proteins with respect to GAPDH or βtubulin after densitometric analysis.
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