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28 protocols using hsp70

1

Western Blot Analysis of Ovarian Tissues

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For western blot analysis, cells and ovarian tissues were collected and lysed with RIPA buffer. The protein concentration of each sample was quantified using a BCA Protein Assay Kit (P0010S, Beyotime, China). Equal amounts of proteins were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto fabrication of polyvinylidene fluoride (PVDF) membranes. The membranes were individually incubated with the specific antibodies, and then incubation with HRP-conjugated secondary antibodies. The enhanced chemiluminescence reagent was used to detect the target proteins. The antibodies used in this study included: PIK3R2 (1:10000, Abcam, USA), SPRED-1 (1:10000, Abcam, USA), calnexin (1:2000, Abcam, USA), AKT (1:2000, Cell Signaling, USA), p-AKT (1:2000, Cell Signaling, USA), ERK1/2 (1:2000, Cell Signaling, USA), p-ERK1/2 (1:2000, Cell Signaling), TSG101 (1:2000, Proteintech, China), HSP70 (1:2000, Proteintech, China), and CD63 (1:1000, Proteintech, China), and GAPDH (1:5000, Proteintech, China).
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2

Protein Expression Analysis in Exosomes

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Total proteins were detached by 10% SDS-PAGE and then transferred onto a polyvinylidene difluoride membrane. After that, the membrane was incubated with primary antibody against BCL6 (1:500, cat. no. #5650, Cell Signaling Technology), integrin αv (1:1000, cat. no. ab179475, Abcam), integrin β3 (1:500, cat. no. ab119992, Abcam), CD63 (1:500, cat. no. AF5117, Affbiotech), CD81 (1:500, cat. no. 27855-1-AP, Proteintech), TSG101 (1:1000, cat. no. 28283-1-AP, Proteintech), HSP70 (1:1000, cat. no. ab2787, Abcam), calnexin (1:1000, cat. no. ab133615, Abcam), Lamp2b (1:500, cat. no. ab18529, Abcam) and β-actin (1:10000, cat. no. TDY051, Bejing TDY Biotech Co., LTD.) at 4°C overnight. After incubating with the HRP-labeled goat anti-rabbit secondary antibody for 1 h at room temperature, protein bands were visualized by the ECL reagents.
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3

Western Blot Analysis of Cellular Proteins

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The cell proteins were extracted using the whole cell lysis assay kit (KeyGen, Nanjing KeyGen Biotech, Nanjing, China), and the protein concentration was measured using BCA protein quantitation assay kit (Nanjing KeyGen Biotech, Nanjing, China). A total of 35 μg protein of each cell sample was separated on a 12% SDS-PAGE gel and then transferred onto the polyvinylidene difluoride membranes. The membranes were fully soaked in western blocking buffer (Beyotime, Beyotime Biotechnology, Shanghai, China) for 60 min and then incubated with BAX (1:2000; Proteintech, Wuhan, China), HSP70 (1:1000; Proteintech, Wuhan, China), GRP78 (1:1000; Proteintech, Wuhan, China), CHOP (1:1000; Proteintech, Wuhan, China), and CYP19A1 (1:1000; Beyotime, Beyotime Biotechnology, Shanghai, China) primary antibody overnight at 4 °C. On the second day, these treated blot membranes were incubated with HRP-conjugated secondary antibody (1:5,000; Beyotime, Beyotime Biotechnology, China) for 60 min at room temperature, respectively. At last, the immunoreactive bands were visualized using a Clarity™ Western ECL Substrate Kit (Bio-Rad Laboratories, Hercules, CA, USA) and were determined by FlourChem HD2 gel imaging and analysis system (ProteinSimple, Santa Clara, CA, USA), and the densitometry of immunoreactive bands was analyzed using Quantity One software 4.6.2 (Bio-Rad Laboratories, Hercules, CA, USA).
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4

Screening of Novel Compounds in Cancer Cell Lines

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B16, HepG2, A549, SW620, MCF-7 and Hela cells were obtained from ATCC and maintained by State Key Laboratory of Biotherapy, Sichuan University in DMEM medium supplemented with 10% fetal bovine serum (FBS, GIBCO, Australia). MTT was purchased from Sigma-Aldrich. Antibodies against hsp90α, stat3 were purchased from Genetex. Antibodies against Bcl-2, Bim, caspase-3, caspase-8, caspase-9, FasL, p27, c-myc, N-cadherin, EGFR, pEGFR(T1068), pEGFR(T1173), C-Raf, pC-Raf, ERK1/2, pERK1/2, Akt, pAkt(S308), pAkt(S473), P90RSK, pP90RSK, Met, pMet, PI3K, B-Raf, pB-Raf, hif1-α were purchased from Cell Signaling Technology. Antibodies against poly polymerase (PARP), FasL, JNK, pJNK, Met, pMet were purchased from Abcam. Antibodies against p21, β-actin, cytochrome c, Bad, Bax, E-cadherin, vimentin, MMP2, MMP9, ZEB1, β-catenin, hsp90β, hsp70, p53, mdm2, cdk2, cdk4, cdk6, cdc37, MNK1, ERK5, GAPDH and Secondary antibodies (HRP-conjugated sheep anti-rabbit antibodies or HRP-conjugated sheep anti-mouse antibodies) for western blot were obtained from Proteintech. Protein A/G Mix Magnetic Beads for Immunoprecipitation were purchased from Millipore. The detailed synthesis, characterization and in vitro biological procedures of compounds 8a–n were described in Supplementary materials.
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5

Quantitative Immunoblotting of Autophagy-Related Proteins

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Cell lysates were collected in 8M urea/0.2% SDS. A BCA assay (Pierce) was used to determine protein concentration. 5 μg of protein was separated on 4–12% bis-tris gradient gels (Invitrogen) and transferred onto nitrocellulose membrane. Revert Total Protein Stain (LI-COR Biosciences) was used to assess protein loading. The following primary antibodies/dilutions were used: BAG3 (Proteintech 10599–1AP, 1:4000), HSP70 (Proteintech 10995–1AP, 1:4000), HSPB8 (Proteintech 15287–1AP, 1:2500), HSPB5 (15808–1AP, 1:2500), SYNPO2 (Proteintech 25453–1AP, 1:2500), LC3 (Cell Signaling Technology 2775s, 1:1000). Secondary antibodies: LICOR IRDye 680RD and 800CW, 1:8000. Membranes were imaged on an Azure c600. Protein densitometry was quantified using ImageStudio (LI-COR Biosciences).
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6

Western Blot Analysis of Signaling Proteins

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Cells and human FLCs were lysed in ice-cold RIPA buffer (10 mM Tris-HCl, 150 mM NaCl, 1% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS, 2 mM EDTA, 50 mM sodium fluoride) with protease inhibitors. Cleared lysate was measured using BCA Protein Assay (Pierce). Lysate was boiled in 1X LDS buffer (Thermo Fisher), separated on 4–12% NuPAGE gradient gels (Thermo Fisher) and transferred onto nitrocellulose using standard techniques. Membranes were incubated overnight at 4°C in 5% w/v milk with TBST and the following antibodies: PKAc (BD Transduction, 610981), Hsp70 (Proteintech, 10995–1), β-actin (Sigma-Aldrich, A1978), AKAP-Lbc (V096, 1 µg ml−1), phospho p44/42 MAPK (CST, 9101), p44/42 (CST, 9102). Membranes were washed in TBST, incubated with HRP-labeled secondary antibodies (GE Life Sciences), washed as before and developed using ECL (Thermo Fisher) on an iBright FL1000. For re-probing, membranes were striped with 1X Re-Blot Plus Strong (Millipore) for 15 min and then re-blocked in Blotto before incubation with primary antibodies again. Densitometry for blot quantification was done using ImageJ software (NIH; http://rsb.info.nih.gov/ij).
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7

Exosome Isolation and Characterization

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Before exosome isolation, the cultured cells were maintained in exosome free medium for 24 h. In brief, 10 ml culture medium with exosome free FBS was collected and centrifuged at 1,000 rpm for 10 min. Then the supernatant was filtrated with 0.22 μm filter to remove the cell debris. Subsequently, the medium containing exosomes was incubated with one quarter volume of PEG (polyethylene glycol) 8000 buffer overnight at 4°C (Sun et al., 2019 (link)). Finally, the mixture was centrifuged at 3,000 rpm for 30 min and re-suspended with 20 μl PBS. The morphology of exosomes was detected by Transmission Electron Microscope (TME), and the particle size distribution was examined via NanoSight. The exosome markers, including HSP70 (Proteintech, No.: 10995-1-AP) and CD63 (Proteintech, No.: 25682-1-AP), were examined by Western blot, and the details of this procedure can be found in our previous study (Mo et al., 2017 (link)).
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8

Exosome Protein Profiling by Western Blot

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Exosomes and cells were lysed with 1 × RIPA lysis buffer (Beyotime, Haimen, China) on ice for 10–15 min. After centrifugation at 12,000 ×g for 5 min at 4°C, total protein extracts were collected and their concentration was detected using the BCA method. The protein (20 μg) was then separated on 10% SDS-polyacrylamide gels and the blots were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% skimmed milk, the membranes were then incubated with primary antibodies at 4°C overnight and secondary antibodies at room temperature for 1 h. The primary antibodies to HSP70, CD81, TSG101, GDF11, and GAPDH (1:2,000, Proteintech, Inc., CA, USA) and corresponding secondary antibodies (anti-rabbit or anti-mouse IgG, 1:5,000, ABclonal, Cambridge, MA, USA) were used. After rinsing, the blots were visualized using the ECL method (Pierce, Thermo Scientific, Waltham, USA) and the protein signals were analyzed using ImageJ software.
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9

Evaluation of Stem Cell Differentiation

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The Sphk2 inhibitor (ABC294640, cat. no. S7174) and 3-methyladenine (3-MA, cat. no. S2767) were obtained from Selleck (Shanghai, China). An AKT inhibitor (LY294002, cat. no. S1737) was obtained from Beyotime (Shanghai, China). Antibodies against GAPDH, IL-6, IL-1, MMP1, p21, p16, Beclin-1, p-AKT, Sphk2, TSG101, HSP70, and Alix were purchased from Proteintech (Wuhan, China). Antibodies against AKT, JNK/p-JNK, and NFκB/p-NFκB were obtained from Abcam (Cambridge, MA, USA). Antibodies against LC3A/B were purchased from KleanAB (Shanghai, China). Antibodies against LC3B (cat. no. bs-4843R) were purchased from Bioss (Beijing, China). Collagenase II (cat. no. A004202) was purchased from Sangon Biotech (Shanghai, China). Tert-butyl hydroperoxide (TBHP, cat. no. 458139),PKH26(cat. no. MINI26), and PKH67(cat. no. PKH67GL) were obtained from Sigma (St. Louis, MO, USA). 1,1′-Dioctadecyl-3,3,3′,3‘-tetramethylindotricarbocyanine iodide (DIR) was obtained from Invitrogen (Carlsbad, CA, USA). MSC osteogenic differentiation medium (cat. no. MUBMX-90021), chondrogenic differentiation medium (cat. no. MUCMX-9004), and adipogenic differentiation medium (cat. no. MUBMX-90031) were provided by Cyagen (Guangzhou, China).
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10

Western Blot Analysis of Extracellular Vesicle Markers

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Extracted protein was separated on 6% or 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels and transferred onto polyvinylidene difluoride membranes (BD Biosciences). Antibodies to BCL6 (1:250 overnight incubation at 4 ℃; CST), TSG101, CD63, ALIX, HSP70 and APOB (1:1000 overnight incubation at 4 ℃; Proteintech) were used with the same secondary antibody horseradish peroxidase (HRP)-labeled goat-anti-rabbit (1:5000; 1 h incubation at room temperature; Proteintech), while β-actin (1:1000 overnight incubation at 4 ℃; Proteintech) was used with the secondary antibody HRP-labeled goat-anti-mouse (1:5000; 1 h incubation at room temperature; Proteintech). Blots were detected by chemiluminescence using the ChemiDoc XRS Imaging System (Bio-Rad).
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