For western blotting analysis, total or nuclear protein was prepared and the concentrations were analyzed using the Bradford method. Samples were loaded on precast 12% SDS‐PAGE gels with approximately 50 µg protein in each lane. The following antibodies and concentrations were used over night at 4°C; ACE (Abcam, ab254222, 1:1000), AT1 (Proteintech, 25343‐1‐AP, 1:1000), AT2 (Proteintech, 13436‐1‐AP, 1:1000), ACE2 (Proteintech, 21115‐1‐AP, 1:1000), MasR (Novus, NBP1‐78444, 1:1000), FOXO1 (Proteintech, 18592‐1‐AP; 1:1000), FOXO3 (Proteintech, 10849‐1‐AP; 1:1000), FOXO6 (Proteintech, 19122‐1‐AP; 1:1000), LC3 (Cell signaling, 4108; 1:1000), ATG5‐ATG12 (Cell signaling, 4180; 1:1000), Beclin‐1 (Abcam, ab62557; 1:1000), NLRP3 (Abcam, ab214185; 1:200), ASC (Novus, NBP1‐78977, 1:1000), caspase‐1 (Abcam, ab1872; 1:1000), IL‐1β (Abcam, ab9722; 1:1000), and β‐actin (Proteintech, 66009‐1‐Ig; 1:4000). Following incubation with the HRP‐conjugated secondary antibody, the signals were detected with an ECL kit (Thermo Fisher) and then quantified using Image J software.
Foxo1
Manufactured by Proteintech
Sourced in China
FOXO1 is a laboratory product offered by Proteintech. It is a protein that plays a key role in regulating cellular processes such as cell cycle progression, apoptosis, and glucose metabolism. The product is intended for use in research applications, but no further details on its intended use or applications are provided.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Proteintech (6 mentions)
Bcl2 associated x (bax), by Proteintech (3 mentions)
Bcl 2, by Proteintech (2 mentions)
Histone h3, by Proteintech (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ecl kit, by Thermo Fisher Scientific (1 mentions)
Ab62557, by Abcam (1 mentions)
Ab1872, by Abcam (1 mentions)
Ab254222, by Proteintech (1 mentions)
Ab9722, by Proteintech (1 mentions)
Nbp1 78444, by Proteintech (1 mentions)
Foxo6, by Proteintech (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Redd1, by Proteintech (1 mentions)
Proliferating cell nuclear antigen (pcna), by Cell Signaling Technology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Phospho foxo1 ser256, by Cell Signaling Technology (1 mentions)
Phospho mtorser2448, by Proteintech (1 mentions)
Cck 8 assay kit, by Beyotime (1 mentions)
11 protocols using foxo1
1
Western Blot Analysis of RAAS and Autophagy Proteins
2
Western Blot Analysis of Cellular Signaling
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Total cell lysates were extracted in RIPA lysis buffer supplemented by protease/phosphatase inhibitor cocktail (KGP2100; KeyGEN BioTECH, Nanjing, China) and incubated for 30 min on ice. The supernatant was collected after centrifugation. The nuclear and cytoplasmic lysates were extracted by using a nuclear and cytoplasmic extraction kit (KGBSP00; KeyGEN BioTECH, Nanjing, China) following the manufacturer's protocol. The protein concentrations were determined by PierceTM BCA Protein Assay Kit (SE248347; Thermo Fisher Scientific). 30 μg of protein from each sample was separated by SDS‐PAGE and transferred to PVDF membranes. The membranes were incubated with the primary antibodies, such as β‐catenin (8480; Cell Signaling Technology), FOXO1 (18592‐1‐AP; Proteintech), phospho‐FOXO1Ser256 (9461; Cell Signaling Technology), REDD1 (10638‐1‐AP; Proteintech), mTOR (67778‐1‐Ig; Proteintech), phospho‐mTORser2448 (67778‐1‐Ig; Proteintech), GAPDH (10494‐1‐AP; Proteintech), and PCNA (13,110; Cell Signaling Technology), at 4°C overnight and subsequently with the secondary antibody conjugated with horseradish peroxidase at room temperature for 1 h. Signals were developed by ECL (MilliporeSigma, Burlington, MA, USA) and visualized by chemiluminescence apparatus. Protein densitometry was quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA).
3
Sweroside Modulates Inflammatory Response
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Sweroside (purity > 98%, Figure 2 A) was obtained from National Institutes for Food and Drug Control (Beijing, China). Fetal bovine serum (FBS) and phosphate buffer saline (PBS) were acquired from Servicebio Company (Wuhan, China). DMEM, DMSO, trypsin, the RIPA Lysis buffer and the PVDF membrane were obtained from Solarbio Company (Beijing, China). The annexin V-FITC apoptosis detection kit was obtained from Vazyme Biotech Company (Nanjing, China). Hoechst 33342, NAM, SIRT1, NF-κB, β-actin and the proliferating cell nuclear antigen (PCNA) antibodies were from Beyotime Institute of Biotechnology (Shanghai, China). The TNF-α, IL-1β, IL-6, IL-10, MnSOD, FOXO1, COX-2 and i-NOS antibodies were obtained from Proteintech Company (Wuhan, China). The ROS assay kit, CCK-8 assay kit, NO assay kit, cell cycle and apoptosis analysis kits were from Beyotime Institute of Biotechnology (Shanghai, China). The PGE2 Elisa Kit was purchased from Hui Jia Company (Xiamen, China). The other chemicals were of analytical grade and were commercially available.
4
Protein Expression Analysis of MC3T3-E1 Cells
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The total protein of MC3T3-E1 cells was extracted using the RIPA buffer supplemented protease inhibitors. The protein samples of each group were separated by electrophoresis on SDS-PAGE and transferred to PVDF membrane. Next, the membrane was blocked using 5% BSA at room temperature for 1 h and subsequently incubated with the primary antibody overnight at 4 °C. The following day, the membrane was washed three times using Tris-buffered saline and incubated with corresponding secondary FITC-conjugated goat antibody for 1 h at 4 °C. Finally, the protein bands were recorded using the Bio-Rad ChemiDoc TM XRS system. All the primary antibodies details were as follows: Bcl2(Cat No: 12789-1-AP), Bax(Cat No: 50599-2-Ig), caspase3(Cat No: 66470-2-Ig), caspase9(Cat No: 66169-1-Ig), GAPDH(Cat No: 60004-1-Ig), Histone-H3(Cat No: 17168-1-AP), PI3K (Cat No: 60225-1-Ig), AKT:(Cat No:10176-2-AP), p-AKT (Cat No: 66444-1-Ig), FOXO1(Cat No: 18592-1-AP) were purchased from Proteintech (Wuhan, China). Phospho-FOXO1 (Catalog No. AF4410) and Phospho-PI3K (Catalog No. AF3417) were purchased from Affinity.
5
Western Blot Analysis of Cell Signaling
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Western blotting was performed for analyses of the expression of FOXO1, CyclinE, CDK2 and P27 in tumor tissues and cell lines as previously described (Zhang et al., 2015 (link)). Primary antibodies against Pan Phospho-Serine/Threonine (Abmart, China), FOXO1ser249, P27Thr187 (Invitrogen, USA), FasL, Bim, TRAIL, GAPDH (Wanleibio, China), FOXO1, CyclinE, CDK2, P27, E2F, Rb (proteintech, USA) were used.
6
Western Blot Analysis of Signaling Proteins
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After extraction of total and nuclear proteins, western blotting was performed with primary antibody against JNK (1:1000, Cell Signaling Technology), phospho-JNK (1:1000, Cell Signaling Technology), ERK1/2 (1:1000, Proteintech), phospho-ERK1/2 (1:1000, Cell Signaling Technology), FOXO1 (1:1000, Proteintech), BAX (1:1000, Proteintech), CASP3 (1:1000, Proteintech), histone H3 (1:5000, Proteintech) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5000, Proteintech) as described previously 16 (link).
7
Western Blot Analysis of Cell Cycle Regulators
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Total protein was extracted using RIPA lysis buffer (Beyotime, Shanghai, China) with a 1% protease inhibitor and phosphatase inhibitor. Twenty-five micrograms of total protein for each sample were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). After blocking in 5% nonfat milk at room temperature for 2 h, the membrane was incubated with primary antibody at 4 °C overnight. The next day, the membrane was incubated with HRP-labeled secondary antibody at room temperature for 2 h. Afterward, signals were visualized by ECL chemiluminescent reagent (Millipore). The antibodies were as follows: FOXO1 (1:1000, Proteintech); p21cip1 (1:2000, Cell Signaling Technology); p27kip1 (1:2000, Cell Signaling Technology); GAPDH (1:3000; Cell Signaling Technology).
8
Western Blot Analysis of TAZ, LATS, Nrf2, Foxo1
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Proteins from uteri or stromal cells were separated by SDS-PAGE and then transferred onto PVDF membranes. After blocking with 5% skim milk, the membranes were probed overnight with antibodies against TAZ (1:1000, Cell Signaling Technology), phospho-TAZ (Ser89, 1:1000, Cell Signaling Technology), phospho-large tumor suppressor kinase (phospho-LATS, Thr1079, 1:1000, Cell Signaling Technology), nuclear factor erythroid 2-related factor 2 (Nrf2, 1:1000, Proteintech), forkhead box O1 (Foxo1, 1:1000, Proteintech) or Gapdh (1:5000, Proteintech) at 4 °C followed by incubation with HRP-conjugated secondary antibody (1:5000). Signals were visualized with an ECL chemiluminescent kit.
9
Western Blot Analysis of Cellular Proteins
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The whole cell lysates were extracted from tissues, Ges-1, AGS, and MKN28 cells using RIPA lysis buffer containing PMSF and phosphatase inhibitor (P0100, Solarbio). Protein content was measured by BCA Protein Detection kit (P0012S, Beyotime Biotechnology, Shanghai, China) to equalize protein loading. After separation by electrophoresis, proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany), which were later blocked using 5% skim milk for 30–60 min. Then, we further incubated PVDF membranes based on DDX56 (1:200; 5A7; sc-101,018, Santa Cruz, CA), Bcl-2 (1:1000, No. 26,593-1-AP, ProteinTech), BAX (1:5000, No. 60,267-1-Ig, ProteinTech), FOXO1 (1:1000, No. 18,592-1-AP, ProteinTech), p21 Cip1 (1:1000, No. 10,355-1-AP, ProteinTech), CDK2 (1:1000, No. 10,122-1-AP, ProteinTech), and GAPDH (1:2000, No. HRP-60004, ProteinTech). After washing with TBST, PVDF membranes were subsequently incubated using HRP-conjugated secondary antibody (ProteinTech) for a 1-h period under ambient temperature. The ECL chemiluminescence kit (Thermo Fisher Scientific, Waltham, MA, USA) was adopted for visualizing blots. Image J (1.8.0, NIH, USA) was utilized for performing image analysis. Protein expression was normalized to GAPDH and the mean value of the group was plotted in a bar chart.
10
Western Blot Analysis of Muscle Protein Expression
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Tissue samples were treated with radioimmunoprecipitation assay (RIPA) buffer containing inhibitors (protease, phosphatase) and centrifuged at 11,500× g at 4 °C for 15 min. The concentration of these protein samples was determined by the bicinchoninic acid protein assay kit (Beyotime, Shanghai, China). Denatured protein samples were isolated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 10% fat-free milk for 2 h and then placed in primary antibodies specific for MyHC-I (Proteintech, Wuhan, China, 22280-1-AP), MyHC-IIA (Abcam, Cambridge, UK, ab124937), MyHC-IIB (Proteintech, Wuhan, China, 20140-1-AP), Akt (Cell Signaling Technology, 4685S), P-Akt (ser473) (Proteintech, Wuhan, China, 66444-1-Ig), FoxO1 (Proteintech, Wuhan, China, 18592-1-AP), P-FoxO1 (ser256) (Signalway Antibody, Greenbelt, MD, USA, 12198), and GAPDH (Proteintech, Wuhan, China, 60004-1-Ig) overnight at 4 °C and finally incubated in secondary antibodies (Proteintech, Wuhan, China, SA00001-1 or SA00001-2) for 1 h at room temperature. Protein signal was visualized by the SmartChemiTM 610 plus (SinSage Technology, Beijing, China).
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