Proteins were extracted from cells using RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer's protocol. Total proteins were separated by SDS-PAGE gel and then transferred into PVDF membranes (Millipore). After incubating with 5% skim milk for 1 h at room temperature, the membranes were incubated with primary antibodies overnight at 4°C. The primary antibodies (Cell Signaling Technology, Beverly, MA., USA) used in this study include anti-Beclin-1 (No. 4122, 1:1000), anti-LC3 I/II (No.12741, 1:1000), anti-p62 (No.88588, 1:1000), anti-FOXM1 (No.5436, 1:1000), anti-p-AKT (No.4060, 1:2000), anti-mTOR (No.2983, 1:1000) and anti-GAPDH (No.5174, 1:2500). Then, the membranes were incubated with corresponding secondary antibodies (Cell Signaling Technology, No.7076 and NO.7074, 1:4000) at room temperature for 1 h. ECL system (Bio-Rad Laboratories) was used for detection of antibody-bound proteins according to manufacturer's instructions.
Anti foxm1
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-FOXM1 is a primary antibody that specifically recognizes the FOXM1 transcription factor. FOXM1 is a key regulator of cell cycle progression and plays a role in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti gapdh, by Cell Signaling Technology (3 mentions)
Anti β catenin, by Cell Signaling Technology (3 mentions)
Anti p62, by Cell Signaling Technology (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Anti sox2, by Cell Signaling Technology (2 mentions)
Anti flag, by Merck Group (2 mentions)
Anti zeb1, by Cell Signaling Technology (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Anti ki67, by Cell Signaling Technology (2 mentions)
Anti stat3, by Cell Signaling Technology (2 mentions)
Anti β actin, by Proteintech (2 mentions)
Anti cxcr4, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti beclin 1, by Cell Signaling Technology (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Anti lc3 1 2, by Cell Signaling Technology (1 mentions)
Anti mtor, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ecl system, by Bio-Rad (1 mentions)
Clarity western ecl blotting substrate, by Bio-Rad (1 mentions)
22 protocols using anti foxm1
1
Western Blot Analysis of Cell Signaling
2
Protein Extraction and Western Blot Analysis
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A proteinase inhibitor cocktail (Sigma, USA) was added to RIPA-lysis buffer (PBS, sodium deoxycholate (0.5%), NP40 (1%), Sodium dodecyl sulphate (SDS, 0.1%) and phenylmethylsulfonylfluoride (100μg/mL)) which was applied to isolate whole tissue or cell protein from surgical samples or cell lines for 30 minutes on ice. The supernatant was harvested from centrifuged lysates. SDS-PAGE was carried out, and the segregated proteins were moved to a fluoride polyvinylidene fluoride (PVDF) membrane. Membranes were blocked for 1 hour at room temperature with 5% powdered skimmed milk, then incubated with primary antibodies overnight at 4°C. The following primary rabbit antibodies were diluted at 1:1000 (Cell Signaling Technology, Massachusetts, USA): anti-Lamin B1, anti-β-catenin and anti-FOXM1. Rabbit anti-GAPDH, also from Cell Signaling, was diluted at 1:3000. Mouse anti-DEPDC1 (1:200) was from Santa Cruz Biotech, Texas, USA). Abcam supplied anti-mouse secondary IgG H&L antibodies labelled with horseradish peroxidase (HRP, diluted 1:2000) incubated with the membrane for one hour. Bio-Rad (California, USA) supplied the Clarity Western ECL Blotting Substrates used to visualize protein locations on the membranes developed with the ChemiDoc XRS+ Bio-Rad imaging system. Our loading controls were GAPDH and Lamin B1.
3
Protein Expression Analysis of CENPU and FOXM1
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Tissues and cells were lysed on ice for 10 minutes in RIPA lysis buffer (Beyotime, Jiangsu, China) including 1% pro tease inhibitor and phenylmethanesulfonyl fluoride (PMSF; Beyotime). The concentration of proteins was quantified using the bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific). Next, 40 µg of total protein from each sample was separated using 10% polyacrylamide gels (Beyotime), which was followed by transfer to polyvinylidene fluoride (PVDF) membranes (Hoffman-La Roche Ltd., Basel, Switzerland). Membranes were subsequently blocked for 1 hour at room temperature with 5% BSA (Boster Biological Technology Co. Ltd., Wuhan, China). The PVDF membranes were, respectively, incubated overnight with mouse monoclonal anti-β-actin (dilution 1:1,000; BOSTER, BM0627), rabbit polyclonal anti-CENPU (dilution 1:500; LS-C158151; LifeSpan BioSciences, Seattle, WA, USA), and rabbit monoclonal anti-FOXM1 (1:500; 20459; Cell Signaling Technology, Danvers, MA, USA). After washing with tris buffered saline tween, membranes were probed with goat anti-rabbit IgG (dilution 1:5,000) or goat anti-mouse IgG (dilution 1:5,000) conjugated with horseradish peroxidase for 1 hour at room temperature. Bands were detected using BeyoECL Plus (Beyotime). Results are expressed relative to β-actin band density, which was used as a loading control.
4
FOXM1 and SOX2 Chromatin Immunoprecipitation
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Cells were fixed with 1% formaldehyde for 10 minutes at room temperature. Sonicated cell lysates were subjected to immunoprecipitation using 5 μg anti-FOXM1 or anti-SOX2 (Cell Signaling Technology, Danvers, MA, USA) or isotype control IgG. Immunoprecipitated DNA was extracted and subjected to polymerase chain reaction analysis. Primers used for ChIP are listed in Table 1 .
5
Protein Isolation and Western Blot Analysis
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Total protein was isolated from cells using cold radio immunoprecipitation assay buffer containing a protease inhibitor cocktail (Roche Applied Science, Penzberg, Bavaria, Germany). A total of 50 μg of protein was subjected to SDS-PAGE, and the blots were transferred onto a polyvinylidene difluoride membrane. After blocking, the membranes were incubated with anti-SIRT1 (Millipore, Temecula, CA, USA), anti-SIRT2 (Cell Signaling Technology, Danvers, MA, USA), anti-SIRT3 (Cell Signaling Technology), anti-SIRT5 (Millipore), anti-SIRT6 (Cell Signaling Technology), anti-eNOS (Cell Signaling Technology), anti-KLF2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-FOXM1 (Cell Signaling Technology), anti-β-actin (Santa Cruz Biotechnology), and anti-FLAG (Sigma-Aldrich) antibodies. Antibodies against cell cycle regulators and checkpoint molecules, including cyclin D1, cyclin D3, p18 INK4C, p21 Waf1/Cip1, p27 Kip1, CDK2, CDK6, phospho-RB, and phospho-p53 (Ser15), were purchased from Cell Signaling Technology. Immune-reactive protein bands were visualized by chemiluminescence using ECL reagents (GE Healthcare, Fairfield, CT, USA). Protein expression was imaged in a ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA, USA).
6
Protein Extraction and Western Blot Analysis
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After extracting the protein with RIPA buffer containing protease inhibitor (Roche), the determination of protein concentrations was performed with an enhanced BCA protein assay kit (Beyotime), followed by Western blotting analysis as described previously (54 (link)). Anti-HA (catalog H6908) was from Sigma-Aldrich. Anti-CKS1 (catalog sc-376663) was purchased from Santa Cruz. Anti-SKP2 (catalog A4046), anti-FN1 (catalog A16678), anti-CCNB1 (catalog A19037), and anti-H3 (catalog A22348) Abs were purchased from Abclonal. Anti-FOXM1 (catalog 20459S), anti-ZEB1 (catalog 83243SF), and anti-GAPDH (catalog 2118S) were purchased from Cell Signaling Technology. Anti-FLAG (catalog 20543-1-AP) and anti-RNF112 Ab (catalog 25165-1-AP) were purchased from Proteintech. Anti-tubulin (catalog AT819) was purchased from Beyotime.
7
Immunoblot Analysis of EMT Markers
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Immunoblot was carried out as described previously.16 (link) Antibodies including anti-MnSOD, anti-FoxM1, anti-E-cadherin, anti-N-cadherin, anti-Twist, anti-MMP-2, anti-Slug, anti-ZEB1 (Cell Signaling Technology, Inc. USA) and anti-β-actin (Sigma, MA, USA) were used as primary antibodies. HRP-conjugated antibodies (Beyotime Institute; China) were used as secondary antibodies. Immunoreactive protein bands were detected using the enhanced chemiluminescence (ECL) reagent (Millipore; USA).
8
Protein-Protein Interaction Assays
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Protein Co-IP assays were performed as described previously. In short, lysates from cultured A549 and HCC827 cells were obtained using RIPA and underwent immunoprecipitation with anti-FOXM1 (1:50, #20459, Cell signaling Technology), anti-β-catenin (1:50, #8480, Cell signaling Technology) or anti-IgG (1:20, #3420, Cell signaling Technology) for 1 h at 37 °C, and protein A-agarose was added for overnight incubation of lysates. Later, complex of protein A-agarose-antigen-antibody was collected using centrifugation for 2 min and underwent wash immunoprecipitation-HAT buffer for 5 times. The binding proteins were tested by western blot.
9
Xenograft Tumor Analysis by IHC and TUNEL
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After pretreatment with ice-cold phosphate buffer saline (PBS) and fixed in 10% buffered neutral formalin (Beijing Solaibao Technology, Beijing, China), tumor xenografts were paraffin-embedded and cut into 4-μm sections. The sections were subjected to IHC staining of Ki67 and FOXM1 expression and TUNEL staining of apoptosis in xenografts after being deparaffinized and rehydrated as described [26 (link)]. For IHC analysis, antigen retrieval was performed using boiling citrate buffer (0.01 M, pH 6.0; Dako, Copenhagen, Denmark), and endogenous peroxidase activity was blocked via 0.3% H2O2. Then, the sections were subjected to incubation with the primary antibody against Ki67 (anti-Ki67; #9449, 1:500 dilution, Cell signaling Technology, Boston, MA, USA) or FOXM1 (anti-FOXM1; #20459, 1:600 dilution, Cell signaling Technology) at 4 °C overnight. After PBS washing, the sections were incubated with secondary antibodies (ab205718, 1:1500 dilution, Abcam, Cambridge, MA, USA) for 1 h at room temperature. After PBS washing, the sections were dyed by diaminobenzidine (DAB, shown in brown). For TUNEL staining, cell apoptosis in above sections was tested by use of In Situ Cell Death Detection Kit (Roche, Mannheim, Germany) as per the manufacturer’s instructions. Images were acquired by a light microscope (Olympus, Tokyo, Japan).
10
Immunohistochemical Analysis of Wound Healing
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Paraffin embedded tissue sections of discarded DFUs, foot skin (FS), oral and skin wounds were used for staining with anti-phospho-STAT3 (1:100; Abcam), anti-MPO (1:1500; Abcam), anti-CD68 (1:800; Abcam), anti-PCNA (1:1000; Cell Signaling), anti-Keratin 5 (1:1000; LSBio), anti-FOXM1 (1:600; Cell Signaling), anti-STAT3 (1:100; Cell Signaling), anti-TNFα (1:25; Abcam), and anti-Ki67 (1:200; Abcam). Murine wounds were excised at day 4 post-wounding and fixed in 4% paraformaldehyde overnight at 4 °C and sections were used for staining with anti-MPO (1:1500; Abcam) and anti-Keratin 5 (1:1000; LSBio). Stainings were visualized with either Alexa Fluor 488-conjugated goat anti-rabbit antibody (1:300; Invitrogen), Alexa Fluor 555-conjugated goat anti-guinea pig antibody (1:300; Invitrogen), Alexa Fluor 647-conjugated goat anti-mouse antibody (1:300; Invitrogen), and mounted with VECTASHIELD antifade mounting media with DAPI (Vectorlabs) to visualize cell nuclei. Specimens were analyzed using a Zeiss LSM 780 confocal microscope and images were acquired with Zen software (Carl Zeiss).
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