Muscle homogenates were separated by SDS-PAGE and transferred onto membranes, as previously described [13 (link)]. Immunoblots were probed by antibodies against GPR30 (1:500, Bioss, Woburn, MA, USA), heat shock protein (HSP) 90 (1:1000, BD Biosciences, San Jose CA, USA), HSP70 (1:1000, BD Biosciences), HSP27 (1:1000, Abcam, Inc., Cambridge, MA, USA), and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:5,000; Cell Signaling, Danvers, MA, USA) was used as a loading control.
Hsp70
Manufactured by BD
Sourced in United States
HSP70 is a protein that functions as a molecular chaperone, assisting in the folding and unfolding of other proteins. It plays a key role in the cellular stress response and protein homeostasis.
Automatically generated - may contain errors
Lab products found in correlation
Hsp90, by BD (3 mentions)
Flag tag (flag), by Merck Group (3 mentions)
P chk1, by Cell Signaling Technology (2 mentions)
Sc 376450, by Santa Cruz Biotechnology (2 mentions)
Snap tag, by New England Biolabs (2 mentions)
Myc tag, by Cell Signaling Technology (2 mentions)
γ tubulin, by Merck Group (2 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Cell Signaling Technology (1 mentions)
Gpr30, by Bioss Antibodies (1 mentions)
Hsp27, by Abcam (1 mentions)
V5 tag, by Thermo Fisher Scientific (1 mentions)
Extrapage one precast gels, by Nacalai Tesque (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Promega (1 mentions)
Supersignal west pico plus, by Thermo Fisher Scientific (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Gapdh ab9485, by Abcam (1 mentions)
Tsg101, by BD (1 mentions)
Goat anti rabbit igg, by ZSGB-BIO (1 mentions)
Calnexin, by BD (1 mentions)
Hlmvec, by Lonza (1 mentions)
9 protocols using hsp70
1
Quantifying Muscle Protein Levels
2
Western Blot Analysis of Protein Samples
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Protein samples were subjected to SDS–polyacrylamide gel electrophoresis (PAGE) on 5–20% ExtraPAGE One Precast Gels (Nacalai Tesque). Membranes were incubated consecutively with primary antibodies and horseradish peroxidase–conjugated secondary antibodies (Promega), and signals were visualized with SuperSignal West Pico PLUS or Dura (Thermo Fisher Scientific) reagents and a ChemiDoc imaging system (Bio-Rad). Primary antibodies included the following: V5 tag (Thermo Fisher Scientific), FLAG tag (Merck), HSP70 (BD Biosciences), and HSP90 (BD Biosciences).
3
Immunoblotting Antibodies for DNA Repair
4
Immunohistochemical Analysis of HSP70 in Frozen Liver
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Frozen liver sections (16 μm thick) were fixed in 1% p-formaldehyde for 10 min at room temperature. After washing with PBS, post-fixation with ethanol:acetic acid 2:1 for 5 min and permeabilization twice with 0.5% Triton X-100 for 15 min were performed. Antibody especific binding was blocked for 1 h in PBS with 10% normal goat serum, 3% bovine serum albumin, 1.5% NaCl and 0.5% Triton X-100 for 1 h. Immunohistochemistry analyses were performed using HSP70 (primary anti-body from BD Science, San Jose, CA, USA) and Alexa 594 Fluor labelled (secondary antibody from Invitrogen, USA), as previously described [8 (link)]. Negative controls were prepared by replacing the first antibody with phosphate saline buffer. Images were obtained with a Nikon Eclipse E1000 fluorescence microscope [8 (link)].
5
Protein Profiling of Microvesicles and Whey Samples
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Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate equal protein amounts of mEVs and whey samples. Proteins were transferred onto polyvinylidene fluoride membranes, blocked with 5% skim milk (BD Biosciences, Franklin Lakes NJ, USA), and detected with the following primary antibodies: HSP70, TSG101, CD63, CD9 and calnexin (cat. no. ab275018, Abcam, UK). After incubation, the membranes were washed by TBST, and then incubated for 50 min at 37 °C with secondary antibody goat anti-rabbit IgG (ZSGB-bio, Beijing, China). The protein bands were washed by TBST and scanned with an automatic ECL image analysis system (Tanon-4800, Shanghai, China). In addition, the antibodies to verify globe proteome were ABAT (ab216465), SCIN (ab199723), DDC (ab131282), ACSL4 (ab155282) and GAPDH (ab9485), purchased from Abcam UK (Abcam, Cambridge, UK).
6
Immunoblotting Antibodies for DNA Repair
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Immunoblotting was performed as described19 (link) with the following antibodies: 53BP1 (175933, Abcam; 1:1000; 100-304, Novus Biological; 1:1000); BRCA1 (MAB22101, R+D systems; 1:500); BRCA2 (OP95, Millipore; 1:500); CHK1 (8408, Santa Cruz; 1:1000); pCHK1 (2341, Cell Signaling Technology; 1:1000); CHK2 (611570, BD; 1:1000); Flag-tag (M2, Sigma; 1:1000); γ-Tubulin (GTU488, Sigma; 1:20,000); GFP (11814460001, Sigma; 1:1000); HA (3724, Cell Signaling Technology; 1:20,000); HSP70 (610608, BD; 1:1000); MAD2L2/REV7 (180579, Abcam; 612266, BD; 1:1000); Myc-tag (9B11, Cell Signaling Technology; 1:1000); OBFC1/STN1 (89250, Abcam; 1:1000; SC-376450, Santa Cruz; 1:1000); PRIM1 (10773-1-AP, Proteintech; 1:1000); RIF1 (#1240, de Lange Lab; 1:1000); SHLD1 (PA5-59280, Thermo-Fisher; 1:1000); SNAP tag (9310, NEB; 1:1000); TRF2 (#1254, de Lange Lab; 1:5,000). Affinity-purified peptide antibodies against mouse SHLD1 and SHLD2 proteins (Chapman Lab, unpublished; 1:1000) were generated by Eurogentec.
7
Cytotoxicity Assay of Pneumolysin on Human Lung Endothelial Cells
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Human lung microvascular endothelial cells (HLMVECs) were obtained from Lonza. Cells were cultured with 5% CO2 at 37°C using EBM-2 MV medium supplemented with EGM-2 MV that was purchased from Lonza. All of the in vitro experiments were performed using passage 3 to passage 5 HLMVECs. PLY was a gift from Dr. Trinad Chakraborty (Institute for Medical Microbiology, Justus-Liebig University, Giessen, Germany) PLY was purified from a recombinant Listeria innocua 6a strain expressing LPS-free PLY. Geranylgeranylacetone (GGA, Sigma) was prepared in DMSO. Tempol, LPS, Glucose, pyruvate, and l -glutamine were obtained from Millipore Sigma. Oligomycin, FCCP, and antimycin were provided in the Seahorse XF Cell Mito Stress Test Kit from Agilent. FITC dextran (Fluorescein isothiocyanate–dextran 4000 and Fluorescein isothiocyanate–dextran 70000) were obtained from Sigma. Antibodies for western blotting included Hsp70 from BD Bioscience, cleaved caspase 3, NF-κB, and GAPDH were from Cell Signaling and Hsp90 from BD Bioscience. Nox1 antibody from Sigma. GFP and Hsp70 adenoviruses were generated in house using established methodologies (38 (link), 39 (link)).
8
Ephrin-B1 and EphB Receptor Imaging
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Mouse embryos were subjected to immunofluorescence using 2 µg/ml ephrin-B1 (AF473; R&D Systems), 2 µg/ml EphB2 (AF467; R&D Systems), 2 µg/ml EphB3 (AF432; R&D Systems), and 10 µg/ml GFP (ab13970; Abcam) antibodies. The following secondary antibodies were used: Alexa Fluor 488–conjugated donkey anti–chicken IgG (1:1,000, 703-545-155; Jackson ImmunoResearch Laboratories, Inc.) and Cy3-conjugated donkey anti–goat IgG (1:400; 705-165-003; Jackson ImmunoResearch Laboratories, Inc.). To label F-actin, Alexa Fluor 647–conjugated phalloidin (1:40; A22287; Thermo Fisher Scientific) was used. For Western blotting, the following primary antibodies were used: rabbit anti-ROCK1 (1:500; sc-5560; Santa Cruz Biotechnology, Inc.), ROCK2 (1:500; sc-5561; Santa Cruz Biotechnology, Inc.), and HSP70 (1:1,000; 610607; BD). The following IRDye secondary antibodies were used: goat anti–mouse 680RD (1:5,000; 925–68070; LI-COR Biosciences) and goat anti–rabbit 800CW (1:5,000; 926–32211; LI-COR Biosciences).
9
Comprehensive Mass Spectrometry Reagents
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All solvents for mass spectrometry analysis, 0.1% formic acid in water and 0.1% formic acid in ACN were of LC-MS grade purchased from EMD (Gibbstown, NJ, USA). Sequencing grade modified trypsin was from Promega (Madison, WI, USA) and Gelcode Blue stain reagent was from Pierce (Rockford, IL, USA). Complete protease inhibitor cocktail tablet was obtained from Roche (Mannheim, Germany). Ammonium bicarbonate, ammonium acetate, DTT, iodoacetamide, Tris-HCl, bromophenol blue, beta-mercaptoethanol, Tween 20, formic acid and SDS were obtained from Sigma-Aldrich (St. Louis, MO, USA). Glycerol was from Life Technologies (Gaithersburg, MD, USA). All buffers and solutions were prepared using deionized water by Milli-Q, Millipore (Bedford, MA, USA). Primary antibodies against human vimentin and heat shock protein-70 (HSP-70) were purchased from BD Biosciences (Franklin Lakes, NJ, USA); those against beta-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz (Santa Cruz, CA, USA). Primary antibody against human beta-catenin was from Cell Signaling (Danvers, MA, USA). Unless stated otherwise, all other chemicals were extra-pure grade or cell culture tested.
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