Cells were lysed in RIPA lysis buffer (R0020, Solarbio) supplemented with phosphatase inhibitor cocktail (4906837001, Roche) and protease inhibitor cocktail (11836170001, Roche). Protein contents were measured with the BCA Protein Assay (23227, Thermo Fisher Scientific). Equal amounts of protein extracts were separated on an SDS–PAGE gel, followed by electrotransfer onto a PVDF membrane (EZWB05-ISEQ00010-1, Millipore). The blots were subsequently incubated for 2 h in a blocking buffer. The membranes were incubated at 4°C overnight with monoclonal antibodies, followed by corresponding secondary anti-rabbit or anti-mouse antibodies for 2 h. The antibodies used for Western blotting were as follows: SLC7A11 (A2413, ABclonal), GPX4 (A1933, ABclonal), ATG5 (10181-2-AP, Proteintech), LC3 (14600-1-AP, Proteintech), β-actin (81115-1-RR, Proteintech), GAPDH (10494-1-AP, Proteintech), TRF2 (22020-1-AP, Proteintech), secondary anti-rabbit antibody (SA00001-2, Proteintech), and anti-mouse antibodies (SA00001-1, Proteintech). The proteins were detected with ECL Blotting Detection Reagents (32106, Thermo Fisher Scientific).
Ecl blotting detection reagents
Manufactured by Thermo Fisher Scientific
Sourced in United States
ECL Blotting Detection Reagents are a chemiluminescent substrate used for the detection of proteins in Western blot analysis. The reagents generate a light signal when exposed to the enzyme-labeled target protein, allowing for sensitive and quantitative detection of the protein of interest.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay, by Thermo Fisher Scientific (4 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Phosphatase inhibitor cocktail, by Roche (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Sa00001 1, by Proteintech (1 mentions)
Sa00001 2, by Proteintech (1 mentions)
γ h2ax, by Abcam (1 mentions)
Dnmt3a, by Cell Signaling Technology (1 mentions)
Dnmt1, by Cell Signaling Technology (1 mentions)
Dnmt3b, by Cell Signaling Technology (1 mentions)
P mtor, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Total protein extraction kit, by Keygen Biotech (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
β actin, by Abcam (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
9 protocols using ecl blotting detection reagents
1
Protein Extraction and Western Blotting
2
Comprehensive Protein Analysis in Mouse Testes
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Total cellular proteins and mouse testicular tissues were extracted using the Total Protein Extraction Kit (Keygentec, China) and protein concentrations were measured using the BCA Protein Assay kit (Pierce, USA). Proteins were run on SDS-PAGE gels, blotted on nitrocellulose membrane, and immunodetected with primary antibodies against PTEN, PI3K, p-PI3K, AKT, p-AKT (Ser473), p-AKT (Thr308), mTOR, p-mTOR (S2448), cleaved caspase-3, Bax, Bcl-2, DNMT1, DNMT3a, DNMT3b (Cell Signaling Technology), and γ.H2AX (Abcam). β-actin (Abcam) were detected as controls. Signals were visualized by ECL blotting detection reagents (Thermo Fisher Scientific, USA) and exposed to X-ray films which were scanned and quantitatively analyzed using Image Lab Software (Bio-Rad, USA).
3
Western Blot Analysis of Protein Expression and Modifications
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Cells were collected, washed, and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer containing Protease and Phosphatase Inhibitor (Thermo Fisher Scientific). A bicinchoninic acid (BCA) kit (CWBio) was then used to detect the concentration of protein. Equivalent amounts of protein were separated on 10% SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore). The membrane was then blocked with Tris-buffered saline with Tween 20 (TBST) buffer containing 5% skim milk powder and incubated with corresponding primary antibodies at 4 °C overnight. The primary antibodies used were anti-p65 (Cell Signaling Technology), anti-IκBα (Cell Signaling Technology), anti-p50 (Cell Signaling Technology), anti-Histone-H3 (Abcam), anti-β-actin (Abcam), anti-Flag (Cell Signaling Technology), GAPDH (Cell Signaling Technology), anti-L-Lactyl Lysine (PTM Bio Inc), anti-Lactyl-Histone H3 (Lys18) Rabbit mAb (PTM Bio Inc), anti-KRAS (Abcam), anti-KRASG12D (Abcam), anti-Lamin A (Abcam). Membranes were then washed with TBST three times and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary antibody (Cell Signaling Technology) for 1 h at room temperature. Signals were developed with ECL Blotting Detection Reagents (Thermo Fisher Scientific).
4
Western Blot Analysis of Cell Signaling Proteins
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H1299 cells were lysed with RIPA buffer (Thermo). Proteins were segregated by SDS-PAGE and then transferred to polyvinylidene fluoride membranes. The membranes were blocked by TBST solution with 5% skim milk and subsequently incubated with primary antibodies against CRKL (1:1000, ab32018, Abcam), p16 (1:5000, 10883-1-AP, Proteintech), p21 (1:2000, 10355–1-AP, 10355–1-AP), and GAPDH (1:2000, 60004-1-Lg, Proteintech). In the end, membranes were incubated with a specific secondary antibody (1:1000, ab205719, Abcam) and visualized by ECL Blotting Detection Reagents (Thermo). Protein quantification was carried out using ImageJ software.
5
Western Blot Protein Analysis
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Total cell lysates were collected in lysis buffer (Bioteke, Beijing, China) supplemented with 1% protease inhibitor and protein concentrations were measured using the BCA method (Thermo). 30–50 μg of whole cell lysates were resolved by SDS-PAGE using 8% or 12% gels and transferred onto PVDF membranes (Roche; Roche Diagnostics, Basel, Switzerland). Then the membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.2% Tween 20 for 1 hour at room temperature. For immunodetection, the membranes were incubated with 1:200–1:5000 diluted primary antibodies overnight at 4 °C and then secondary antibodies conjugated with horseradish peroxidase for 1 h at room temperature. The signals were visualized by ECL blotting detection reagents (Thermo) and exposed to X-ray films which were scanned and quantitatively analyzed by Quantity One (Bio-Rad, California, USA). ACTB was used as corresponding loading control.
6
Exosomal Surface Marker Detection
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The surface markers CD9 and TSG101 of exosomes were detected using Western Blot (WB). Briefly, 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel was used to separate 20 µg protein extract; after which, a semi-dry transfer system was employed for relocation to a PVDF membrane. Next, 5% evaporated skimmed milk containing TBS-Tween 20 (0.05%) was used for blocking at room temperature for 2 h, followed by an overnight incubation at 4 °C on a membrane with primary antibodies against CD9 (ab92726, Abcam, Cambridge, UK) and TSG101 (ab125011, Abcam, Cambridge, UK). Next, the membranes were incubated for 2 h with HRP-coupled secondary antibodies (Feiyi Biotech, Wuhan, China). Photographic film and ECL blotting detection reagents were employed for protein band visualization (Thermo, Waltham, MA, USA).
The exosomal surface marker CD63 was analyzed by flow cytometry (FCM) analysis with vesicles pre-absorbed on latex beads (4% polystyrene latex beads, ThermoFisher, USA). The bead-exosome aggregates were labeled with the fluorescence-labeled antibodies anti-CD63 (1:200 eBioscience).
The exosomal surface marker CD63 was analyzed by flow cytometry (FCM) analysis with vesicles pre-absorbed on latex beads (4% polystyrene latex beads, ThermoFisher, USA). The bead-exosome aggregates were labeled with the fluorescence-labeled antibodies anti-CD63 (1:200 eBioscience).
7
Whole Cell Protein Extraction and Western Blot
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Cells were lysed in ice-cold whole cell extract buffer (50 mM TRIS-HCl, pH 8.0, 4 M urea and 1% Triton X-100), supplemented with complete protease inhibitor mixture (Roche Diagnostics, 04693132001). The whole cell extracts were resolved by SDS-PAGE gel electrophoresis, and transferred to nitrocellulose membranes. Membranes were probed with the indicated primary antibodies followed by appropriate HRP-conjugated secondary antibodies (KPL). Protein bands were visualized using ECL Blotting Detection Reagents (Thermo Scientific, 32106). Primary antibodies used for western blotting were as follows: Parkin (Cell Signaling, 4211), LC3B (Sigma, L7543), MFN1 (Abcam, ab57602), TIM23 (Proteintech, 11123-1-AP), COXIV (Cell Signaling, 4844), C-III core 1 (Invitrogen, 459140), p62 (MBL, PM045), cleaved Caspase-3 (Cell signaling Technology, 9664), cleaved PARP (Cell signaling Technology, 5625), Actin (Proteintech, 60008-1-IG), α-Tubulin (Sigma, T9026).
8
Western Blot Analysis of Cell Signaling Proteins
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Cells were lysed in RIPA lysis buffer supplemented with phosphatase inhibitor cocktail and protease inhibitor cocktail (Roche). Protein content was measured with the BCA Protein Assay (Thermo Fisher Scientific). Equal amounts of protein extracts were separated on the SDS-PAGE gel, followed by electrotransfer onto a PVDF membrane (Millipore). The blots were subsequently incubated for 2 h in blocking buffer (5% nonfat milk in TBST). The membranes were incubated in 4°C overnight with monoclonal antibodies and followed by corresponding secondary anti-rabbit or anti-mouse antibodies for 2 h. The antibodies used for Western blot were as follows: Beta-actin (ProteinTech), TERT (ABclonal), mTOR (ABclonal), p-mTOR (Ser2448, Cell Signaling Technology), BENC1 (Santa Cruz Biotechnology), p62 (Cell Signaling Technology), LC3 (Abcam), and secondary anti-rabbit or anti-mouse antibodies (ProteinTech). The protein was detected with the ECL Blotting Detection Reagents (Thermo Fisher Scientific).
9
Gyp-L Protein Expression Analysis
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Cells treated with Gyp-L in the presence or absence of inhibitors for indicated times were harvested and lysed in RIPA buffer (Beyotime, P0013B) containing 1 mM PMSF. The lysates were normalized to equal amounts of protein, and were separated by 6–15% gradient SDS-PAGE. After transferring to nitrocellulose membrane, the proteins were probed with the indicated primary antibodies. Detection was conducted by incubation with species-specific HRP-conjugated secondary antibodies. Immunoreactive bands were visualized using ECL blotting detection reagents (Thermo, 34080). GAPDH was used as the loading control.
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