Extraction of total protein was made from the lung cells using RIPA (Solarbio, Beijing, China) buffer and centrifuged at 12,000× g. Cell protein was separated by 10% SDS-PAGE, and then transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 10% skimmed milk (P0216, Beyotime, Shanghai, China) and incubated with primary antibodies. The monoclonal antibody was obtained from Abcam (Cambridge, MA, USA). HRP conjugated AffiniPure goat anti-rabbit IgG (H + L) was obtained from Proteintech (Proteintech Group, Rosemont, IL, USA). Detailed antibody information is in the supplementary file .
P0216
Manufactured by Beyotime
Sourced in China
The P0216 is a laboratory centrifuge designed for general-purpose applications. It features a maximum speed of 6,000 RPM and a maximum capacity of 4 x 100 mL tubes. The centrifuge is equipped with a brushless motor and an electronic speed control system to ensure consistent and reliable performance.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (3 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Ipvh00010, by Merck Group (2 mentions)
Radioimmunoprecipitation assay (ripa), by Solarbio (1 mentions)
Hrp conjugated affinipure goat anti rabbit igg h l, by Proteintech (1 mentions)
Ipvh20200, by Merck Group (1 mentions)
P0018fm, by Beyotime (1 mentions)
P1005, by Beyotime (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Nd 2000, by Thermo Fisher Scientific (1 mentions)
Ab284130, by Abcam (1 mentions)
Imagej 5, by Bio-Rad (1 mentions)
β actin antibody, by Proteintech (1 mentions)
Vcam 1 antibody, by Santa Cruz Biotechnology (1 mentions)
P0012ac, by Beyotime (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Rock1, by Abcam (1 mentions)
Cyclin d1, by Abcam (1 mentions)
β catenin, by Abcam (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
15 protocols using p0216
1
Western Blot Analysis of Lung Cells
2
Western Blot Analysis of Osteogenic Markers
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In the presence of a protease inhibitor (Beyotime, China, P1005), the osteogenic hFOB 1.19 cells were lysed using RIPA lysis buffer (Beyotime, China, P0013B), and centrifugation at 4 °C (12,000 rpm) for 15 min collected the supernatant. Protein samples were separated by SDS–PAGE electrophoresis and then transferred to polyvinylidene fluoride (PVDF; Millipore, IPVH20200) membranes. The TBST containing 5% skim milk (Beyotime, China, P0216) was used to block the nonspecific binding site for 1 h, and the antibodies were incubated overnight at 4 °C. After incubation with the antimouse IgG (Jackson, 115-035-003) or antirabbit IgG (Jackson, 111-035-003) at room temperature for 1 h, the signal was detected with an enhanced chemiluminescence reagent (Beyotime, China, P0018FM). Table 2 presents the list of the primary antibodies.
The primary antibody information of this study
| Primary antibody name | Company and catalog | Dilution | Molecular weight (kDa) |
|---|---|---|---|
| RUNX2 | CST, 8486 | 0.736111111 | 55–62 |
| OPN | abcom,ab8448 | 0.736111111 | 66 |
| OCN | Abcam, ab133612 | 0.736111111 | 11 |
| GAPDH | Proteintech 60,004–1-Ig | 5.597222222 | 36 |
3
RNA Isolation and m6A Quantification
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All processes were based on an existing protocol [28 ]. For this assay, total RNA from PTECs (5 × 106) was isolated using TriZol (15596-026, Invitrogen, USA), incubated at room temperature for 5 min. Thereafter, polyadenylated mRNA was purified from the extracted total RNA. The concentration of RNA was determined using a spectrophotometer (ND-2000, ThermoFisher, USA), and the quantity of RNA sample was checked via electrophoresis on a 0.8% agarose gel (16500-500, Invitrogen, USA). Next, RNA samples were prepared and loaded with the wells. The wells were then covered with foil to prevent light, and electrophoresis was run at 35 V overnight.
For the m6A content quantification test, after confirming that the RNA samples were transferred from the gel to the membrane, the membrane was blocked in 5% nonfat milk (P0216, Beyotime, China) in TBST at room temperature for 1 h, rinsed in TBST for 15 min, and incubated with the primary antibody (Abcam, UK) against m6A (ab284130) or IgG (ab172730) at 4 °C overnight and with the secondary antibody against rabbit IgG at room temperature for 1 h. Further, BeyoECL Plus reagent was applied to the membrane, and the m6A content was calculated using ImageJ 5.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
For the m6A content quantification test, after confirming that the RNA samples were transferred from the gel to the membrane, the membrane was blocked in 5% nonfat milk (P0216, Beyotime, China) in TBST at room temperature for 1 h, rinsed in TBST for 15 min, and incubated with the primary antibody (Abcam, UK) against m6A (ab284130) or IgG (ab172730) at 4 °C overnight and with the secondary antibody against rabbit IgG at room temperature for 1 h. Further, BeyoECL Plus reagent was applied to the membrane, and the m6A content was calculated using ImageJ 5.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
4
Protein Separation and Detection Protocol
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The protein samples were separated using SDS-PAGE (Beyotime, P0012AC, Shanghai, China). The stacking gel was run at 70 V, and the separating gel at 120 V. The proteins were transferred to a PVDF membrane (Merk Milipore, IPVH00010, MA, USA) at a constant voltage of 110 V for 80 min. After blocking with 5% skimmed milk solution (Beyotime, P0216, Shanghai, China) for 1 h, the membrane was incubated with β-actin antibody (Proteintech, 66009, Rosemont, IL, USA) at 1:20000 dilution and VCAM-1 antibody (Santa Cruz, sc-18864, Dallas, TX, USA) at a 1:1000 dilution overnight at 4 °C. After washing, a secondary antibody solution at a 1:20,000 dilution was added and incubated at room temperature for 2 h. Finally, a chemiluminescence kit (Beyotime, P0018S, Shanghai, China) was used to visualize the protein bands using a gel imaging instrument (Tanon, Tanon-5200, Shanghai, China).
5
Western Blot Analysis of Protein Signaling
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Whole protein lysates of the indicated samples were extracted by RIPA lysis buffer (JRDUN, Shanghai, China) with ethylene diamine tetraacetic acid (EDTA)-free protease inhibitor cocktail (Roche, Mannheim, Germany). The protein concentration was estimated by using an enhanced bicinchoninic acid protein assay kit (Thermo Fisher, Cleveland, OH, USA). Equal amounts of total protein (25 μg) were fractionated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA) overnight. After being blocked with 5% nonfat dry milk (P0216, Beyotime, Shanghai, China) for 1 h at room temperature, the membranes were probed at 4 °C overnight with the primary antibodies (ROCK1 [1:2000; Abcam, Cambridge, UK], ROCK2 [1:1000; Abcam, UK], DLC-1 [1:1000; Abcam, UK], β-catenin [1:5000; Abcam, UK], cyclin D1 [1:10,000; Abcam, UK], and GAPDH [1:2000; Cell Signaling Technology, Beverly, MA, USA]), and then the secondary anti-mouse IgG antibody (1:1000; Beyotime, Shanghai, China) for 1 h at 37 °C. An enhanced chemiluminescence system (Tanon, Guangzhou, China) was used to detect the protein expression value. The protein levels were normalized to GAPDH.
6
Western Blot Analysis of Autophagy Proteins
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The cellular proteins were extracted form skeletal muscle cells and lysed by RIPA lysate (R0010, Solarbio, Peking, China). Equal amounts of protein were electrophoresed on sodium lauryl sulfate polyacrylamide gel (SDS-PAGE) with a loading of 20 μL per well. The protein was then transferred to an activated PVDF membrane (FFP26, Beyotime, Shanghai, China) and blocked with 5% skim milk powder (P0216, Beyotime, Shanghai, China) for 2 h. Appropriately diluted primary antibodies against Beclin-1 (Abcam, Cat# ab223372), p62 (Abcam, Cat# ab101266), p-mTOR (Abcam, Cat# ab109268), mTOR (Abcam, Cat# ab2732), p-AKTSer473 (Abcam, Cat# ab18206), p-IRS1Ser616 (CST, Cat# 2386S), IRS (CST, Cat# 2382S), AKT (CST, Cat# 2938S), LC3 (Affinity, Cat# AF5402) and β-actin (Abcam, Cat# ab8227) were added and incubated at 4 °C overnight. After washing the membrane, the corresponding secondary antibody (Cat# A0208 and A0216, Beyotime, Shanghai, China) was added for incubation. Chemiluminescence detection was then carried out using an enhanced chemiluminescence reagent (P0018AS, Beyotime, Shanghai, China). After development and fixing treatment, the film was photographed by a gel imaging analysis system (UniCel Dxl800, Beckman Coulter, Pasadena, CA, USA).
7
Western Blot Analysis of Cellular Proteins
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Total proteins and nuclear/cytoplasmic proteins were extracted with RIPA lysis buffer (P0013B, Beyotime, Shanghai, China) and a Nuclear-Cytoplasmic Extraction Kit (P0028, Beyotime, Shanghai, China), respectively. Protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (88518, Thermo Fisher Scientific, Waltham, MA, USA). The membranes were blocked with TBST (ST673, Beyotime, Shanghai, China) containing 5% skim milk (P0216, Beyotime, Shanghai, China) for 1 hr at room temperature and then incubated with appropriate antibodies at 4 °C overnight. After washing, the membranes were incubated with HRP-conjugated anti-IgG (Proteintech, Wuhan, Hubei, China) for 2 h at room temperature. The antigen-antibody complexes on the membranes were detected with High-sig ECL reagent (180-5001, Tanon, Shanghai, China). The images were acquired using the MicroChemi system (70-25-00, NDR Bio-Imaging Systems, Jerusalem, Israel) and quantified with ImageJ software (ImageJ 1.50i, National Institutes of Health, Bethesda, MD, USA). GAPDH and Histone H3 served as internal controls. The antibodies used in this study are listed in Supplementary Table S3 .
8
Western Blot Analysis of Hypoxia Markers
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The cell lysate was prepared in ice-cold RIPA buffer (P0013C, Beyotime Biotechnol ogy, Shanghai, China), in which cocktail protease inhibitor (Thermo Fisher Scientific, Shanghai, China) was freshly added. The proteins were separated in homemade SDS-PAGE gel and transferred onto PVDF membrane. Blocking was performed with 5% non-fat milk (P0216, Beyotime Biotechnology, Shanghai, China) in TBST (Tris-buffered saline + 0.1% Tween 20). After blocking, the membrane was incubated with primary antibody overnight and washed with TBST four times, and then incubated with HRP-linked secondary antibody (Beyotime Biotechnology, Shanghai, China) for 2–3 h and washed with TBST. The signal was developed with WesternBright ECL HRP substrate (Advansta, Menlo Park, CA, USA) and captured using a chemiluminescent imaging system (Tanon 5200). Primary antibodies used in this study included HIF1a (ab179483, Abcam, Cambridge, UK), ANGPTL4 (18374-1-AP, Proteintech, Wuhan, China), ATP6AP1 (15305-1-AP, Proteintech, Wuhan, China), CA12 (15180-1-AP, Proteintech, Wuhan, China), and a-Tubulin (T6199, Sigma, Shanghai, China).
9
Western Blotting Analysis of Protein Expression
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The cells were lysed using RIPA lysis buffer (P0013B; Beyotime) for total protein extraction. The proteins were quantified using a BCA kit (P0012; Beyotime), separated by 10% SDS-PAGE (P0012A; Beyotime), and transferred onto 0.45-μm polyvinylidene fluoride (PVDF) membranes (IPVH00010; Millipore, Billerica, USA). The membranes were blocked with 5% nonfat milk (P0216; Beyotime) for 2 h at room temperature and then incubated with primary antibodies against α-SMA (1:1000, bs-10196R; Bioss, Beijing, China), SM22α (1:1000, 10493-1-AP; ProteinTech, Rosemont, USA), CNN1 (1:1000, bs-0095R; Bioss), OPN (1:1000, bs-0026R; Bioss), Collagen I (1:1000, 14695-1-AP; ProteinTech) and β-actin (1:2000, 20536-1-AP; ProteinTech) overnight at 4°C. The membranes were then incubated with goat anti-rabbit IgG (1:5000, SA00001-2; ProteinTech) at 37°C for 1 h. An ECL chemiluminescence kit (P0018S; Beyotime Institute of Biotechnology) was utilized for chemiluminescence detection with the Bio-Rad Gel Doc XR
+ Gel Imaging System (Bio-Rad, Hercules, USA). β-Actin served as an internal control. ImageJ 2x software was used to quantify the protein bands.
+ Gel Imaging System (Bio-Rad, Hercules, USA). β-Actin served as an internal control. ImageJ 2x software was used to quantify the protein bands.
10
Western Blot Assay for Mitochondrial Proteins
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For western blot assay, cells were seeded in a six‐well plate and incubated with Oxa, ART, and OART (2 μM) for 24 h. After the end of the experiment in vivo, the tumor tissue was collected and stored at −80°C. Then, all the samples both cell and tumor tissue were extracted and homogenized by RIPA lysis buffer. The lysates were centrifuged at 13000 g for 10 min at 4°C. Protein extracts were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred to 45 μm polyvinylidene difluoride (Millipore) membranes. Blocked with 5% nonfat milk (P0216; Beyotime) in TBS‐0.1%Tween 20 (TBST) for 2 h at room temperature. The membranes were then incubated with diluent primary antibodies overnight at 4°C. Antibodies and working diluted concentrations were GAPDH (Abcam; ab215191, 1:10,000), DHODH (Proteintech; 14877‐1‐AP, 1:1000), Tom20 (CST; 42406, 1:1000), OPA1 (CST; 80471, 1:1000), MFF (CST; 84580, 1:1000), and GPX4 (Abcam; ab125066, 1:5000).
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