Total protein was extracted from tissues and cells with radio-immunoprecipitation assay lysis buffer (P0013B, Beyotime) containing 100 mM phenylmethylsulphonyl fluoride. The nuclear and cytoplasmic protein was extracted with kit (P0028, Beyotime) according to the instructions, followed by measurement of protein concentration with a bicinchoninic acid kit (P0011, Beyotime). After separation by 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein was transferred onto a polyvinylidene fluoride membrane (1620177, Bio-Rad). Next, the membrane was blocked with 5% skimmed milk powder for 1 h and underwent overnight incubation at 4 °C with primary rabbit antibodies against β-actin (4970, 1:5000, Cell Signaling Technology, Beverly, MA), TGF-β (ab215715, 1:1000, Abcam, Cambridge, UK) and SREBP2 (ab228653, 1:1000, Abcam). The following day, the membrane was incubated with horseradish peroxidase-labeled secondary antibody goat anti-rabbit IgG (ab6721, 1:5000, Abcam) at room temperature for 1 h. Afterwards, the immunocomplexes on the membrane were visualized using enhanced chemiluminescence reagent (1705062, Bio-Rad). Image exposure was performed on Image Quant LAS 4000 C gel imager (GE company, Fairfield, CT). With β-actin serving as the internal reference, protein expression was quantified utilizing the Image J software.
P0011
Manufactured by Beyotime
Sourced in China
P0011 is a general-purpose laboratory centrifuge designed for basic separation and sedimentation processes. It features a fixed-angle rotor capable of accommodating various sample tube sizes. The centrifuge provides adjustable speed settings and a timer function for controlled operation.
Automatically generated - may contain errors
Lab products found in correlation
P0013, by Beyotime (4 mentions)
P0018, by Beyotime (3 mentions)
A0208, by Beyotime (3 mentions)
Ab6721, by Abcam (2 mentions)
Ipvh00010, by Merck Group (2 mentions)
A0216, by Beyotime (2 mentions)
St506, by Beyotime (2 mentions)
P0027, by Beyotime (2 mentions)
P0028, by Beyotime (1 mentions)
Ab215715, by Abcam (1 mentions)
Ab228653, by Abcam (1 mentions)
Protein quantification kit, by Beyotime (1 mentions)
Ab38449, by Abcam (1 mentions)
Ab76198, by Abcam (1 mentions)
Ab76199, by Abcam (1 mentions)
Ab6789, by Abcam (1 mentions)
Ab8805, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
St505, by Beyotime (1 mentions)
Ecl luminescence kit, by Solarbio (1 mentions)
20 protocols using p0011
1
Protein Extraction and Western Blot Analysis
2
Western Blotting Quantification Protocol
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Western blotting was performed as previously described [20 ]. Proteins were collected using RIPA buffer (P0013B, Beyotime, China), protease inhibitors (p1005, Beyotime, China), and phenylmethylsulfonyl fluoride (ST505, Beyotime, China), quantified by a protein quantification kit (P0011, Beyotime, China), and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, electrophoresis was conducted to transfer the proteins into a PVDF membrane (160-0184, Bio-Rad, USA). The membrane was blocked by 5% dried skimmed milk powder for 1 h and then incubated with antibodies against Akt (1: 500, 56kDa, ab8805, Abcam, UK), phosphorylated (p)-Akt (1: 1000, 56kDa, ab38449, Abcam, UK), endothelial nitric oxide synthase (eNOS) (1: 1000, 133kDa, ab76198, Abcam, UK), p-eNOS (1: 500, 140kDa, ab76199, Abcam, UK), and GAPDH (1: 1000, 36kDa, ab8245, Abcam, UK). After incubation for 24 h, the membrane was washed by 1% Tris-buffered saline with Tween 20 and incubated with goat anti-rabbit secondary antibody (1: 10 000, ab205718, Abcam, UK) or goat anti-mouse secondary antibody (1: 10000, ab6789, Abcam, UK). An ECL luminescence kit (PE0010, Solarbio, China) and a gel imaging system (FluorChem FC3, Alpha, USA) were used to expose the membrane and visualize the protein bands, respectively. ImageJ2x (Rawak Software, Germany) was used to analyze the results.
3
Protein Extraction and Western Blot Analysis
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Samples were homogenized in cell lysis buffer (P0013, Beyotime) supplemented with the protease inhibitor phenylmethylsulfonyl fluoride (1:100, ST507, Beyotime) and centrifuged at 12000 × g for 30 min. The supernatant was collected, and the total protein concentration was determined using a BCA protein assay kit (P0011, Beyotime). Equal amounts of protein were separated on 4–12% bis‐Tris gels with MOPs running buffer at 140 V for 1.5 h and then transferred to nitrocellulose membranes using a Trans‐Blot system at 100 V for 1 h. Then, the membranes were washed with 1 × TBST for 5 min at room temperature (20 °C), shaken, and blocked with 5% skim milk in 1 × TBST for 1 h at room temperature. The following antibodies were diluted in 1 × TBST as indicated: 1:3000 for anti‐LC3 (14600‐1‐AP, Proteintech), 1:3000 for anti‐P62 (18420‐1‐AP, Proteintech), 1:10 000 for anti‐mouse IgG (A9044, Sigma‐Aldrich), 1:10 000 for anti‐rabbit IgG (A0545, Sigma‐Aldrich), 1:2000 for anti‐DNALI1 (ab155490, Abcam & 17601‐1‐AP, Proteintech); 1:1000 for anti‐Tau5 (ab80579, Abcam); 1:1000 for Anti‐Phospho‐Tau (Ser202, Thr205) (MN1020, Thermo Fisher Scientific). All uncropped images of western blots are shown in Figure S9 (Supporting Information).
4
Triglyceride Quantification in Drosophila
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The level of triglyceride (TAG) was quantified using Triglyceride (TG) Content Assay Kit (geruisi, G0910W), following the manufacturer instructions. In brief, 5 beheaded females with indicated phenotypes or treatments were homogenized in 200 ul TG extracts. Homogenized samples were centrifuged at 12000 rpm for 10 min at room temperature, 20 ul of the supernatant extract were used to measure triglycerides, and 10 ul were for quantification of protein concentrations (P0011, Beyotime Biotechnology, China) according to the manufacturer instructions. Normalized TAG levels to protein levels. Note: The kit measures the sum of all kinds of TAGs. 9 replicates for each genotype were carried out. Starvation lasts 72 h in adults and 32 h in larvae of all starvation-associated assays.
5
HUVECs Protein Expression Analysis
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HUVECs were lysed on ice for 30 min with the cell lysate. The protein concentration was measured using the BCA method (P0011, Beyotime, Shanghai, China) before being subjected to electrophoresis and transferred to a PVDF membrane (IPVH00010, Millipore, Germany). The PVDF membrane was incubated with antibodies against p-HSF1, HSF1, VEGF, p-Akt, Akt, and GAPDH overnight at 4 °C and finally exposed and photographed. The antibody information was listed in Supplementary Table S1 .
6
Isolation and Characterization of Exosomes from Cell Culture
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Exosomes were obtained from culture medium as previously described with some modification44 . In brief, cells were grown in RPMI 1640 media supplemented with 10% exosome-free FBS. Next, the media was collected and centrifuged at 300×g for 10 min to pellet cells, followed by a centrifugation step of 2000×g for 15 min to discard dead cells, and a centrifugation of 10,000×g for 30 min to discard cell debris. The supernatant was filtered through a 0.22 μm pore filter (syringe filter, SLGP033RS, Millipore, USA). This was followed by two ultracentrifugations at 100,000×g for 70 min to pellet the exosomes. All spins were performed at 4 °C.
Exosomes were resuspended by PBS (02-024-1A, Biological Industries, Israel) and then examined by Electron Microscopy using negative staining. Exosomes used for protein immunoblotting were resuspended in PBS, quantified by bicinchoninic acid assays (P0011, Beyotime) and mixed with SDS loading buffer (C516031, Sangon Biotech, China). Exosomes used for size distribution were resuspended and diluted in PBS, then analysed by ZetaView (Particle Metrix, Germany). Exosomes used for RNA extraction were resuspended in 700 μL of QIAzol Lysis Reagent (79306, QIAGEN) and processed as the protocol of miRNeasy Micro Kit (217084, QIAGEN).
Exosomes were resuspended by PBS (02-024-1A, Biological Industries, Israel) and then examined by Electron Microscopy using negative staining. Exosomes used for protein immunoblotting were resuspended in PBS, quantified by bicinchoninic acid assays (P0011, Beyotime) and mixed with SDS loading buffer (C516031, Sangon Biotech, China). Exosomes used for size distribution were resuspended and diluted in PBS, then analysed by ZetaView (Particle Metrix, Germany). Exosomes used for RNA extraction were resuspended in 700 μL of QIAzol Lysis Reagent (79306, QIAGEN) and processed as the protocol of miRNeasy Micro Kit (217084, QIAGEN).
7
Western Blot Protein Detection Protocol
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Tissues were lysed with 1 mL RIPA lysis buffer and supplemented 1% protease inhibitor cocktail (Solarbio) for 2–5 min with steel balls in a tissue lyser (Qiagen, Germany). Tissue lysates were incubated with the buffer at 4 °C overnight (cell samples were incubated in lysis buffer for 30 min at 4 °C). The lysate was centrifuged at 12,000 rpm for 10 min, the supernatant was collected, and the protein concentration was determined using the BCA method (P0011, Beyotime Biotechnology). The supernatant was mixed with loading buffer and boiled at 100 °C for 10 min. Samples were loaded and subjected to 10% SDS-PAGE. After transfer to PVDF membranes, blots were blocked with 5% skimmed milk powder and incubated with primary antibody overnight at 4 °C. The primary antibodies included a BATF antibody (8638 S, Cell Signaling Technology, USA), anti-β-actin (GB11001, Servicebio, China), and β-tublin (15115, Cell Signaling Technology). Secondary antibody was goat anti-rabbit (Jackson, 111-035-003), images were acquired using the BIO-RAD protein gel imaging system (Universal hood II), and band intensity was calculated using Image J (Zhang et al., 2021c (link)).
8
Western Blot Analysis of Protein Expression
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Total protein lysates were extracted with cell lysis buffer (P0013, Beyotime) containing a protease inhibitor (ST506, Beyotime) and quantified with a BCA assay kit (P0011, Beyotime). Protein samples were loaded on SDS‒PAGE gels and transferred onto polyvinylidene difluoride membranes. After being blocked in 5% BSA for 1 h at room temperature, the membranes were hybridized with primary antibodies against JMJD1C (1:1000; A20153, ABclonal, Shanghai, China), HK2 (1:1000; A0994, ABclonal), PGK1 (1:1000; A12686, ABclonal), LDHA (1:1000; A0861, ABclonal), p-STAT3 (1:1000; AF3293, Affinity), STAT3 (1:1000; AF6294, Affinity), cyclin D1 (1:1000; A19038, ABclonal), CDK4 (1:1000; A11136, ABclonal) or β-actin (1:1000; sc-47778, Santa Cruz, Dallas, TX, USA) overnight at 4 °C, followed by goat anti-rabbit IgG (1:5000; A0208, Beyotime) or goat anti-mouse IgG (1:5000; A0216, Beyotime) secondary antibody incubation for 45 min at 37 °C. The protein signals were visualized by an enhanced chemiluminescence system (P0018, Beyotime) and quantified using Gel-Pro-Analyzer Software (WD-9413B, Beijing Liuyi Biotechnology Co., Ltd., Beijing, China).
9
Antioxidant Biomarkers in Rat Hippocampus
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The substantia nigra (125 mm3 in size) was taken from the hippocampus of rats with different treatment. A total of 1 mL phosphate buffer saline (PBS) homogenate was added to the substantial nigra, followed by centrifugation at 12 000 × g at 42°C for 10 minutes. Following extraction of the supernatant, the protein concentration was measured using a bicinchoninic acid detection kit (P0011, Beyotime) and the content of MDA (A003‐1‐2), SOD (A001‐3‐2) and GSH (A006‐2‐1) in the hippocampus was determined using the MDA, SOD and GSH kit (Nanjing Jiancheng Bioengineering Institute.
Neurons were inoculated into 6‐well culture plates at a cell density of 6 × 104 cells/mL. The contents of SOD, MDA and GSH were measured according to kits’ instructions (Nanjing Jiancheng Bioengineering Institute).
Neurons were inoculated into 6‐well culture plates at a cell density of 6 × 104 cells/mL. The contents of SOD, MDA and GSH were measured according to kits’ instructions (Nanjing Jiancheng Bioengineering Institute).
10
Mouse Colonic Tissue Protein Analysis
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Mouse colonic tissues and ECs were lysed using the radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with a mixture of protease inhibitors to obtain protein samples, and samples were centrifugated and the protein concentration was examined by a bicinchoninic acid kit (P0011, Beyotime). Equal amounts of proteins were received sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes, and after blockade with defatted milk, the membranes were incubated with primary antibodies against GPX4 (1:1000, ab125066, Abcam), IRF7 (1:1000, ab288440, Abcam), SLC11A2 (1:1000, ab55735, Abcam), and β-actin (1:5000, ab6276, Abcam) and with appropriate amount of horse radish peroxidase-conjugated secondary antibody IgG (1:1000, ab6721, Abcam). Protein signals were visualized using the enhanced-chemiluminescence reagent (P0018, Beyotime).
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