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Anti hsp70

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Anti-Hsp70 is a laboratory reagent that detects the presence of the heat shock protein 70 (Hsp70) in biological samples. Hsp70 is a highly conserved molecular chaperone protein that plays a crucial role in cellular stress response and protein folding. The Anti-Hsp70 reagent can be used to measure the levels of Hsp70 in various experimental and diagnostic applications.

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Lab products found in correlation

Anti cleaved caspase 3, by Cell Signaling Technology (3 mentions) Anti poly adp ribose polymerase parp, by Cell Signaling Technology (2 mentions) Anti atp5a, by Abcam (2 mentions) Restore plus western blot stripping buffer, by Thermo Fisher Scientific (2 mentions) Pseudokinase mlkl phospho s345, by Abcam (2 mentions) Nitrocellulose membrane, by Bio-Rad (2 mentions) Anti gapdh, by Abcam (2 mentions) Anti cleaved caspase 7, by Cell Signaling Technology (2 mentions) Anti h3k36me3, by Abcam (1 mentions) Anti h3k4me3, by Merck Group (1 mentions) Anti egfr, by Santa Cruz Biotechnology (1 mentions) Anti phospho egfr, by Cell Signaling Technology (1 mentions) Anti idh1, by Cell Signaling Technology (1 mentions) Anti akt, by Cell Signaling Technology (1 mentions) Sds page, by Thermo Fisher Scientific (1 mentions) Anti phospho akt, by Cell Signaling Technology (1 mentions) Hybond pvdfmembranes, by Cytiva (1 mentions) Anti me1, by Abcam (1 mentions) Anti histone h3, by Cell Signaling Technology (1 mentions) Anti h3k9me3, by Active Motif (1 mentions)

7 protocols using anti hsp70

1

Western Blot Analysis of Protein Markers

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Total protein from each sample (10–20 μg) was separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). Abs used for protein detection were the following: anti–poly(ADP-ribose) polymerase (PARP) (product no. 9542S; Cell Signaling Technology, Danvers, MA), anti–mixed lineage kinase domain-like pseudokinase (MLKL) phospho-S345 (product no. ab196436; Abcam), anti-GAPDH (product no. ab181602; Abcam), anti-HSP70 (product no. 610607; BD Biosciences), and anti-ATP5a (product no. ab176569; Abcam). Western blot membranes were stripped of Abs using Restore Plus Western Blot Stripping Buffer (Thermo Fisher Scientific) when additional Ab probing was required.
Zhu M., Barbas A.S., Lin L., Scheuermann U., Bishawi M, & Brennan T.V. (2018). Mitochondria Released by Apoptotic Cell Death Initiate Innate Immune Responses. ImmunoHorizons, 2(11), 384-397.
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2

Quantitative Western Blot Analysis

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For all Western Blot analyses, proteins were separated by
4–12% SDS/PAGE (Life Technologies), transferred to Hybond PVDF
membranes (Amersham), blocked with 5% milk in PBS with 0.1%
Tween 20 (PBS/Tween) for 1 hr, and incubated with the following antibodies:
anti-cleaved caspase-3 (Cell Signaling, 9664), anti-cleaved caspase-7 (Cell
Signaling, 9491), anti-Hsp70 (BD Pharmingen, 610607), anti-IDH1 (Cell Signaling,
8137), anti-H3K4me3 (Millipore, CS200580), anti-H3K9me3 (Active Motif, 39161),
anti-H3K27me3 (Millipore, CS200603), anti-H3K36me3 (Abcam, ab9050), anti-Histone
H3 (Cell Signaling, 4499), anti-ME1 (Abcam, ab97445), anti-FoxO6 (Thermo
Scientific PA5–35117), anti-phospho-FoxO6 (Abcam, ab154832),
anti-phospho-EGFR (Cell Signaling, 2236S), anti-EGFR (Santa Cruz, sc-373746),
anti-phospho-Akt (Cell Signaling, 4060S), and anti-Akt (Cell Signaling, 9272S).
The blots were subsequently incubated with goat anti-rabbit IgG or goat
anti-mouse IgG antibodies (Santa Cruz) in 5% milk in PBS/Tween and
developed with Supersignal West Dura ECL (enhanced chemiluminescence) substrate
(Pierce) following manufacturer’s protocol. Quantification of blots was
determined by densitometry using ImageJ software.
Calvert A.E., Chalastanis A., Wu Y., Hurley L.A., Kouri F.M., Bi Y., Kachman M., May J.L., Bartom E., Hua Y., Mishra R.K., Schiltz G.E., Dubrovskyi O., Mazar A.P., Peter M.E., Zheng H., James C.D., Burant C.F., Chandel N.S., Davuluri R.V., Horbinski C, & Stegh A.H. (2017). Cancer-associated IDH1 promotes growth and resistance to targeted therapies in the absence of mutation. Cell reports, 19(9), 1858-1873.
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3

Western Blot Analysis of Apoptosis Markers

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Samples were run on NuPAGE 4 to 12% bis-tris gels (Life Technologies) and transferred to Hybond P polyvinylidene difluoride membranes (Genesee Scientific, GE). Membranes were washed for 5 min in PBS + 0.05% Tween 20 (PBS-T), blocked with 5% milk in PBS-T for 1 hour, incubated with primary antibodies overnight, washed three times with PBS-T for 10 min per wash, and developed with secondary goat anti-rabbit, goat anti-mouse, or donkey anti-goat IgG antibodies (Santa Cruz Biotechnology) in 5% milk PBS-T. After three more washes for 10 min each with PBS-T, membranes were developed with SuperSignal enhanced chemiluminescence (ECL) (Thermo Fisher Scientific) following the manufacturer’s protocol. The following primary antibodies were used: anti–cleaved caspase-3 (9664, Cell Signaling), anti–cleaved caspase-7 (9491, Cell Signaling), anti-IDH3α (SAB100035, Sigma-Aldrich), anti-Hsp70 (610607, BD Biosciences), anti-cSHMT (ab186130, Abcam), anti-H3 (4499S, Cell Signaling), and anti-COXIV (4850S, Cell Signaling).
May J.L., Kouri F.M., Hurley L.A., Liu J., Tommasini-Ghelfi S., Ji Y., Gao P., Calvert A.E., Lee A., Chandel N.S., Davuluri R.V., Horbinski C.M., Locasale J.W, & Stegh A.H. (2019). IDH3α regulates one-carbon metabolism in glioblastoma. Science Advances, 5(1), eaat0456.
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4

EV Protein Characterization by Western Blot

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Cell lysates and EV protein samples were denatured in sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer by heating at 95 °C for 15 min. Criterion 4–20% TGX Precast Gels from Bio-Rad was used to separate the proteins and blotted as previously described. Blots were incubated with the primary antibodies, anti-Alix (Abcam, Cambridge, MA), anti-tumor susceptibility gene 101 protein (TSG101, Abcam), anti-CD63 (Santa Cruz, Santa Cruz, CA), anti-HSP70 (BD, San Jose, CA), and anticleaved caspase-3 (Cell Signaling, Danvers, MA), followed by goat or rat anti-Ig secondary antibodies. Specific bands were detected using enhanced chemiluminescent substrate from GE Healthcare (Piscataway, NJ) and visualized on the ImageQuant LAS 4000 imaging system from GE Healthcare. For total EV protein, Ponceau S red staining was used for loading control. β-actin was used as an internal control for cell lysates. Relative band intensity was measured densitometrically using ImageJ software.
Cobbs A., Chen X., Zhang Y., George J., Huang M.B., Bond V., Thompson W, & Zhao X. (2019). Saturated fatty acid stimulates production of extracellular vesicles by renal tubular epithelial cells. Molecular and cellular biochemistry, 458(1-2), 113-124.
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5

Western Blot Analysis of Protein Markers

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Total protein from each sample (10–20 μg) was separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). Abs used for protein detection were the following: anti–poly(ADP-ribose) polymerase (PARP) (product no. 9542S; Cell Signaling Technology, Danvers, MA), anti–mixed lineage kinase domain-like pseudokinase (MLKL) phospho-S345 (product no. ab196436; Abcam), anti-GAPDH (product no. ab181602; Abcam), anti-HSP70 (product no. 610607; BD Biosciences), and anti-ATP5a (product no. ab176569; Abcam). Western blot membranes were stripped of Abs using Restore Plus Western Blot Stripping Buffer (Thermo Fisher Scientific) when additional Ab probing was required.
Zhu M., Barbas A.S., Lin L., Scheuermann U., Bishawi M, & Brennan T.V. (2018). Mitochondria Released by Apoptotic Cell Death Initiate Innate Immune Responses. ImmunoHorizons, 2(11), 384-397.
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6

Characterization of Extracellular Vesicle Protein Markers

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MP and EXO pellets containing 6 µg of protein were lysed with Laemmli sample buffer and run on 10% gels. Platelet and K562 cell lysates were used as positive controls. Proteins were transferred onto Protran nitrocellulose membrane (Whatman International Ltd., Kent, UK). Membranes were blocked with 5% (w/v) skim milk powder (Valio, Helsinki, Finland) in TBS-T and probed with primary mouse anti-CD41 (Beckman Coulter, Inc.), anti-CD9 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-TSG101 (BD Transduction Laboratories) and anti-Hsp70 (BD Pharmingen) for 1 h at RT in 3% milk in TBS-T followed by incubation with the secondary antibody anti-mouse-IgG-HRP (GE Healthcare Limited, Buckinghamshire, UK) in 3% milk in TBS-T for 45 min at RT. After extensive washing, the blots were incubated 1 min at RT with Pierce ECL Western Blotting Substrate (Thermo Scientific) and visualized with Amersham HyperfilmTM ECL (GE Healthcare Limited).
Aatonen M.T., Öhman T., Nyman T.A., Laitinen S., Grönholm M, & Siljander P.R. (2014). Isolation and characterization of platelet-derived extracellular vesicles. Journal of Extracellular Vesicles, 3, 10.3402/jev.v3.24692.
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7

Comprehensive Protein Expression Analysis

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All chemicals were purchased from Sigma. Anti-TRAP1, anti-Hsp90, anti-Cyt c, anti-Hsp70, anti-Drp1, and anti-Bax antibodies were purchased from BD Biosciences; anti-Grp94, anti-Akt, anti-Chk1, anti-c-FLIP, anti-HER2, anti-Sorcin, and anti-SDHB antibodies were purchased from Santa Cruz Biotechnology; anti-PHB, anti-pS9-GSK3β, anti-GSK3β, anti-COX-2, anti-HSF1, anti-SIRT3, and anti-calcineurin A antibodies were purchased from Cell Signaling Technology; an anti-calcineurin regulatory subunit B antibody was purchased from Sigma; an anti-CypD antibody was purchased from Thermo; and an anti-β-actin antibody was purchased from MP Biomedicals.
Park H.K., Yoon N.G., Lee J.E., Hu S., Yoon S., Kim S.Y., Hong J.H., Nam D., Chae Y.C., Park J.B, & Kang B.H. (2020). Unleashing the full potential of Hsp90 inhibitors as cancer therapeutics through simultaneous inactivation of Hsp90, Grp94, and TRAP1. Experimental & Molecular Medicine, 52(1), 79-91.
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