Total protein was isolated from tissue samples using RIPA buffer (Sigma-Aldrich). After quantification using Bradford assay(Thermo Fisher Scientific, Waltham, United States), 20 μg of total protein were pretreated with loading buffer and loaded in 5–20% SDS-polyacrylamide gel (Bio-Rad Laboratories, Hercules, United States). After migration, the samples were transferred into a nitrocellulose membrane (GE Healthcare Life Science). Subsequently, the membranes were blocked using 5% skim milk in PBS buffer for 1 h. Further, the membranes were incubated overnight at 4°C in the primary antibody diluted in 1% skim milk in PBS buffer. The primary antibodies used were anti-SIRT2 antibody (Cell Signaling Technology, Beverly, CA), anti-TFEB antibody (Abcam, Cambridge, United Kingdom), anti-TSG101 antibody (Abcam, Cambridge, United Kingdom), anti-CD63 antibody (Abcam, Cambridge, United Kingdom), anti-HSP70 antibody (Abcam, Cambridge, United Kingdom), and anti-LC3 antibody (Cell Signaling Technology, Beverly, CA). For housekeeping, anti-β-actin antibody (Sigma, St. Louis, MO) was used. After washes with PBS, the membranes were incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA) and were visualized using Odyssey Imaging Systems (LI-COR Biotechnology, Lincoln, United Kingdom).
Anti hsp70 antibody
Manufactured by Abcam
Sourced in United Kingdom
Anti-HSP70 antibody is a laboratory reagent that can be used to detect the presence of the HSP70 protein, which is a member of the heat shock protein family. HSP70 plays a role in protein folding and the cellular stress response. This antibody can be used in various immunoassay techniques to measure or visualize the expression of HSP70 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd63 antibody, by Abcam (3 mentions)
Anti tsg101 antibody, by Abcam (2 mentions)
Fitc labeled secondary anti rabbit antibody, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Sds polyacrylamide gel, by Bio-Rad (1 mentions)
Bradford assay, by Thermo Fisher Scientific (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Anti lc3 antibody, by Cell Signaling Technology (1 mentions)
Anti sirt2 antibody, by Cell Signaling Technology (1 mentions)
Anti tfeb antibody, by Abcam (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Electrophoretic transfer cell, by Bio-Rad (1 mentions)
Anti cd81 antibody, by Abcam (1 mentions)
Gradient polyacrylamide gel, by Bio-Rad (1 mentions)
Sheep anti mouse igg conjugated to hrp, by Neobioscience (1 mentions)
Quality one software, by Bio-Rad (1 mentions)
Anti collagen 1 antibody, by Abcam (1 mentions)
9 protocols using anti hsp70 antibody
1
Western Blot Analysis of Protein Markers
2
Hyperthermia-induced Hsp70 expression
3
Protein Expression Analysis by Western Blot
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Sodium dodecyl sulfate (SDS) sample buffer (0.5 M Tris-HCl, pH 6.8, 20% sucrose, 10% SDS, 1% bromophenol blue) was added to the concentrated samples. After equal amounts of protein (5 μg/lane) were separated on 4–20% gradient polyacrylamide gels (Bio-Rad, Hercules, CA, USA) under nonreducing conditions, the gels were blotted onto nitrocellulose membranes (Pierce, Waltham, MA, USA) in an electrophoretic transfer cell (Bio-Rad). Blots were blocked with 5% non-fat milk at 4 °C overnight and then probed with Anti-Collagen I antibody (1:1000; Abcam, Cambridge, UK, ab23446), anti-CD63 antibody (1:1000; Abcam, ab59479), anti-Hsp70 antibody (1:1000; Abcam, ab2787) and anti-CD81 antibody (1:1000; Abcam, ab59477) at room temperature for 1 h. After three washes with PBS-T (pH 7.4 and 0.1% Triton), the membrane was incubated with sheep anti-mouse IgG conjugated to HRP (NeoBioscience, Shenzhen, China) for 1 h at room temperature, followed by three washes in PBS-T. The band density was determined by Bio-Rad Quality One Software.
4
Immunohistochemistry and TUNEL Assay for Tissue Analysis
5
Immunofluorescence Analysis of Hsp70 Expression
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Immunofluorescence
experiments against heat shock protein 70 (Hsp70) were performed to
evaluate an increase of its expression as a consequence of the treatment
with Ang-LMNVs + AMF stimulus. U87 MG cells were seeded (15 ×
103 cells/cm2) at the center of WillCo dishes
(1 cm2 total area) and then incubated with 325 μg/mL
of Ang-LMNVs in HEPES-supplemented complete medium for 2 days before
being stimulated for 2 h with the AMF, as previously described. Negative
controls and positive controls were also performed; in particular,
negative controls were represented by cultures without any treatment,
while positive controls were placed for 1 and 2 h in an incubator
at 42 °C. This temperature was chosen since it is the typical
temperature achieved during hyperthermia treatments. After the stimulation,
the samples were placed at 37 °C for a further hour in the incubator.
Afterward, the samples were fixed with 4% of PFA at 4 °C. Immunostaining
was performed with anti-Hsp70 antibody (1:50, Abcam) for 2 h at 37
°C; after three washing steps, FITC-labeled secondary anti-rabbit
antibody (1:250, Invitrogen) was added and incubated for 1 h. Nuclei
staining was performed with Hoechst 33342 (1:1000 dilution, Invitrogen)
for 20 min; images were acquired with the confocal microscope.
experiments against heat shock protein 70 (Hsp70) were performed to
evaluate an increase of its expression as a consequence of the treatment
with Ang-LMNVs + AMF stimulus. U87 MG cells were seeded (15 ×
103 cells/cm2) at the center of WillCo dishes
(1 cm2 total area) and then incubated with 325 μg/mL
of Ang-LMNVs in HEPES-supplemented complete medium for 2 days before
being stimulated for 2 h with the AMF, as previously described. Negative
controls and positive controls were also performed; in particular,
negative controls were represented by cultures without any treatment,
while positive controls were placed for 1 and 2 h in an incubator
at 42 °C. This temperature was chosen since it is the typical
temperature achieved during hyperthermia treatments. After the stimulation,
the samples were placed at 37 °C for a further hour in the incubator.
Afterward, the samples were fixed with 4% of PFA at 4 °C. Immunostaining
was performed with anti-Hsp70 antibody (1:50, Abcam) for 2 h at 37
°C; after three washing steps, FITC-labeled secondary anti-rabbit
antibody (1:250, Invitrogen) was added and incubated for 1 h. Nuclei
staining was performed with Hoechst 33342 (1:1000 dilution, Invitrogen)
for 20 min; images were acquired with the confocal microscope.
6
Quantifying Leaf Proteins and HSP70
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Total proteins from rice leaf tissues were extracted with P-PER® Plant Protein Extraction Kit (Pierce, Cat # 89803). Proteins were fractionated by SDS-PAGE and the HSP70 protein levels were detected by western blotting for detection using an anti-HSP70 antibody (Abcam, Cat # ab5439). The Rubisco large subunit, which was detected with a Rubisco-specific antibody (Agrisera, Cat # AS07 259), was used an loading control. The antigen-antibody complexes were detected by enhanced chemiluminescence using luminal as substrate. The western blot films were scanned, and the intensities of protein bands were quantified using the Image J software. The quantification reflected the relative amounts as a ratio of each HSP protein band to the lane’s Rubisco loading control.
7
Extracellular Vesicle Protein Analysis
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Cells and EVs were lysed with NP40 lysis buffer (Thermo Fisher Scientific), and the protein concentration was determined via a BCA assay (Beyotime). Immunoblotting was performed following the standard protocol with a Bio-Rad system using the following primary antibodies: CD63 monoclonal antibody (Ts63) (Thermo Fisher Scientific), CD9 monoclonal antibody (Thermo Fisher Scientific), CD81 monoclonal antibody (M38) (Thermo Fisher Scientific), anti-Hsp70 antibody (Abcam), and calnexin rabbit pAb (ABclonal). The secondary antibodies included an anti-mouse IgG HRP-linked antibody (Cell Signaling Technology), and an anti-rabbit IgG HRP-linked antibody (Cell Signaling Technology). The signal was developed by an enhanced chemiluminescent (ECL) kit (Merck).
8
Protein Expression Analysis in Cells
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The cells of each group were lysed in lysis buffer. Determine the quality of the harvested protein by used BCA kit. Then, 20 μg of total proteins was separated in SDS-PAGE gels and transferred to PVDF membrane. The membranes were blocked for 1 h at room temperature and incubated overnight at 4 °C with the relevant antibodies: anti-ALP antibody (1:1000, Abcam, USA), anti-OCN antibody (1:1000, Abcam, USA), anti-RUNX2 antibody (1:1000, Abcam, USA), anti-SLIT2 antibody (1:1000, Abcam, USA), anti-CD63 antibody (1:1000, Abcam, USA), anti-TSG101 antibody (1:1000, Abcam, USA), anti-HSP70 antibody (1:1000, Abcam, USA) and anti-GAPDH antibody (1:10,000, Abcam, USA). Membranes were rinsed and incubated for 1 h with secondary antibodies (Abcam, USA). After three times of washing, membranes were exposed with the ECL kit. Bands were analyzed using ImageJ software (version 1.6 NIH) to analyze the relative expression levels of the above markers.
9
Cell Signaling Pathway Inhibition
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Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Hyclone (GE Healthcare Life Sciences, Logan, UT, USA). The anti-HSP70 antibody was purchased from Abcam (Cambridge, UK). The anti-Caspase-3, anti-Bcl-2, anti-Bax, anti-ERK, anti-p-ERK, anti-AKT, anti-p-AKT, anti-GAPDH and anti-β-Actin antibodies, as well as LY294002 (the PI3K/AKT inhibitor) and U0126 (the MAPK/ERK inhibitor) were all purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).
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