The total proteins were prepared, and their concentrations were detected using a Total Protein Extraction Kit (Solarbio, Beijing, China). Cell lysates were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare, USA). Then, 5% non-fat milk in TBST buffer was used for the blockage of membranes containing protein. The primary antibodies used in this study were: anti-HSP70 (1: 1000, Abcam, ab79852), anti-hnRNPA2B1 (1: 1000, Abcam, ab31645), and anti-CD9 (1: 1000, Abcam, ab92726). HRP-conjugated secondary goat anti-mouse (1: 5000, Proteintech, Rosemont, IL, SA00001-1) or goat anti-rabbit (1: 5000, Proteintech, SA00001-2) antibodies were incubated for 2 h at room temperature. The relative grey values of immunoreactive bands were calculated based on GAPDH.
Ab79852
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab79852 is a laboratory product offered by Abcam. It is a specific antibody designed for use in research applications. The core function of this product is to detect and bind to a target molecule, enabling researchers to study its properties and behavior. Detailed information about the intended use or specific applications of this product is not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab134045, by Abcam (3 mentions)
Ab92726, by Abcam (2 mentions)
Sa00001 2, by Proteintech (2 mentions)
Ab223052, by Abcam (2 mentions)
Ab6721, by Abcam (2 mentions)
Ab109201, by Abcam (2 mentions)
Ab32503, by Abcam (2 mentions)
Ab194583, by Abcam (2 mentions)
Chemiluminescence detection kit, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab205718, by Abcam (2 mentions)
Ab216130, by Abcam (2 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Ab31645, by Abcam (1 mentions)
Total protein extraction kit, by Solarbio (1 mentions)
Sa00001 1, by Proteintech (1 mentions)
Bm101, by Biomiga (1 mentions)
Gel pro analyzer 4, by Media Cybernetics (1 mentions)
Ab185966, by Abcam (1 mentions)
17 protocols using ab79852
1
Western Blot Protein Analysis
2
Exosome Protein Expression Analysis
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Cells (1 × 106) were lysed with lysis buffer. Supernatant was collected followed by the detection of protein concentration using BCA (PC0020, Beijing Solarbio Science and Technology Co., Ltd, Beijing, China). The isolated 50 µg proteins were separated by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis, and then transferred to a nitrocellulose membrane (LC2005, Thermo Fisher Scientific, Waltham, MA, USA). The membrane was then blocked for 2 h with skim milk powder and probed with primary antibody of rabbit polyclonal antibodies to CD63 (ab134045), CD9 (ab223052), CD81 (ab155760), HSP70 (ab79852), SOX9 (ab185966) and GAPDH (ab181612) from (Abcam, Cambridge, UK) at 4°C overnight. The goat anti-rabbit immunoglobulin G (IgG; ab97051, Abcam, Cambridge, UK) labelled by horseradish peroxidase (HRP) served as the secondary antibody for 1 h of incubation at a room temperature. The membrane was then immersed in enhanced chemiluminescence reaction solution (BM101, Biomiga, San Diego, CA, USA) for development for 1 min in a dark room. The image was subjected to grey scale analysis using Gel-Pro Analyzer 4.0 (Media Cybernetics, Bethesda, MD, USA). The protein expression was normalized to endogenous GAPDH.
3
Spinal Cord Injury Immunohistochemistry Analysis
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Spinal cords were collected 14 days after injury and samples were quickly frozen at −40°C immersed in 4% paraformaldehyde. Transverse 10 µm thick sections of spinal cord were used for immunohistochemistry analysis. Following permeabilization in 0.25% Triton X-100/PBS for 10 min at room temperature, sections were blocked with 10% goat serum (cat. no. SL038; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 60 min at room temperature. Sections were stained using anti-nuclear factor-κB (NF-κB) p65 (1:2,000; cat. no. ab16502; Abcam) and anti-HSP70 (1:100; ab79852; Abcam) at 4°C overnight. Samples were incubated with secondary HRP-conjugated goat anti-rabbit antibody (1:10,000; cat. no. ab205718; Abcam) at 37°C for 1 h. The DAB Horseradish Peroxidase Color Development kit (cat. no. P0202; Beyotime Institute of Biotechnology) was used for signal development (sections were incubated at room temperature for 25 min). Then, 3 fields were randomly selected for each sample, and the positive area in each field was collected and quantified using ImageJ software version 1.32 (National Institutes of Health).
4
Western Blot Analysis of Hsp70 Protein
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Total protein was extracted from the tissues, and the protein concentration was measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Then, 40 μg of each sample was loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, Hercules, CA, USA). After separation, the proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Burlington, MA, USA), and blocked with 5% dried skimmed milk powder for one hour. The membranes were incubated with the primary rabbit polyclonal antibodies to Hsp70 (1: 10000) (ab79852; Abcam, Cambridge, UK) and β-actin (1: 1000) (ab8227; Abcam, Cambridge, UK) overnight at 4°C. The membrane was washed three times using TBST, containing TBS and 1 ml/L of Tween-20, for 10 mins. The membranes were then incubated with goat anti-rabbit IgG (1: 2000) (ab6721; Abcam, Cambridge, UK) for one hour at room temperature. After washing, the results were detected using enhanced chemiluminescence (ECL) and quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Protein expression levels were normalized to β-actin.
5
Exosome Isolation from hAD-MSCs
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After hAD-MSCs reached 90% confluency, the cells were washed and serum-starved for 6 h in basal medium (DMEM/F-12; Gibco). Thereafter, the medium was collected and centrifuged at 720 ×g for 30 min at 4℃ to remove cellular debris, followed by centrifugation at 100,000 ×g for 100 min at 4℃ (Sorvall LYNX-6000, Thermo Fisher Scientific) to sediment the EXOs. The protein content of the concentrated EXOs was measured using the bicinchoninic acid (BCA) protein assay kit (CW0014; CWBiotech, Beijing, China). The morphology of the exosomes was observed using transmission electron microscopy (TEM; JEOL-1200-EX II; JEOL, Tokyo, Japan). Exosomes were identified using specific antibodies targeting CD63 (ab217345, Abcam), CD9 (ab92726, Abcam), CD81 (ab109201, Abcam), and heat shock protein 70 (Hsp70; ab79852, Abcam), which were used for western blotting analysis. All antibodies were diluted to 1：1,000.
6
Histological Evaluation of Bone Formation
7
Mitochondrial Protein Extraction and Western Blot Analysis
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Proteins from cells or lungs were extracted using RIPA Lysis Buffer with Complete Protease Inhibitor Cocktail (Cell Signaling, Danvers, MA, USA). To detect the release of AIF from mitochondria, cells or tissue mitochondria isolation kit (Beyotime, Shanghai, China) were used to separate the protein, and cytoplasm (without mitochondria fractions) was collected for WB. WB was performed as described previously [23 (link)]. The protein levels were measured with specific primary antibodies including HSP70 (ab79852, Abcam, Cambridge, UK), KANK2 (PA5-116620, Invitrogen, Waltham, MA, USA), Bax (ab32503, Abcam, Cambridge, UK), Bcl-2 (ab194583, Abcam, Cambridge, UK), GAPDH (9485, Abcam, Cambridge, UK), β-actin (ab8226, Abcam, Cambridge, UK), and AIF (4642, Cell Signaling, Danvers, MA, USA) at a dilution of 1:1000. The appropriate secondary antibodies were chosen to incubate with membrane for 2 h at room temperature. Finally, protein bands were applied with enhanced chemiluminescence detection kit (Thermo Scientific, Portsmouth, NH, USA) and imaged by fusion-capture software (Fusion FX7, Marne-la-Vallée, France).
8
Western Blot Analysis of Kidney Injury Markers
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Both kidney tissue and HK-2 cells were lysed in RIPA lysis buffer containing protease inhibitor cocktail. Protein concentrations were measured using a BCA protein assay kit (Invitrogen, USA). Protein samples in 10% sodium dodecyl sulfate-polyacrylamide difluoride (SDS-PAGE) membranes were separated and then transferred to polyvinylidene fluoride membranes (PVDF) (Millipore, MA). After blocking by 5% non-fat milk at RT for 1 h, membranes were incubated with primary antibodies against Hsp70.1 antibody (1:1000; PA5-97846, ThermoFisher), HSP70 (1:1000; ab79852, Abcam), TRAF6 (1:1000; ab137452, Abcam), GAPDH (1:1000; 9485, Abcam), IκBα (1:1000; ab32518, Abcam), p-IκBα (1:1000; ab133462, Abcam), p65 (1:1000; ab32536, Abcam), p-p65 (1:1000; ab222494, Abcam), TNF-α (1:1000; ab183218, Abcam), IL-6 (1:1000; ab259341, Abcam), Bax (1:1000; ab32503, Abcam), Bcl2 (1:1000; ab194583, Abcam), caspase 3 (1:1000; ab184787, Abcam) overnight at 4 °C. After washing with Tris-Buffered Saline Tween-20 (TBST) buffer on the next day, the membranes were incubated in secondary antibodies (Goat Anti-Rabbit IgG, 1:5000, ab205718, Abcam) at RT for 2 h. At last, protein bands were treated with chemiluminescence detection kit (Thermo Fisher Scientific, USA) and detected by Fusion-capture system (Fusion, France).
9
Immunohistochemical Analysis of HSP Proteins
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Sections were labeled for HSP27 (Abcam, Mouse ab79868, 1:200 dilution), HSP70 (Abcam, Rabbit ab79852, 1:200 dilution) and HSP90 (Abcam, Mouse ab13492, 1:200 dilution) using the streptavidin/peroxidase complex immunostaining technique, respectively. Primary antibodies were incubated for 2 hours at 37°C. The reaction products were formed with diaminobenzidine. Nuclear counterstaining was performed with hematoxylin. Negative controls were obtained by omitting the first-layer antibody.
10
Quantification of Apoptosis and Stress Markers
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The cleaved caspase-3, HMGB1, or HSP70-positive cells were determined by immunohistochemical staining as previously described.16 17 (link) Briefly, the frozen sections of tumor tissue from KCKO-Luc bearing mice were stained with primary antibodies including cleaved caspase-3 (#9664S, Cell Signaling Technology), anti-HMGB1 antibody (ab18256, Abcam), or anti-HSP70 antibody (ab79852, Abcam), followed by horseradish peroxidase-labeled secondary antibody staining. DAB (3,3′-Diaminobenzidine) was applied as substrate and hematoxylin as counterstain. Positive cells were enumerated using a computerized Olympus DP80 imaging system. Ten randomly selected fields (×400, magnification) of each tumor tissue section were enumerated, and the means were reported.
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