Cells and tissue samples were lysed in ice-cold radioimmunoprecipitation assay buffer supplemented with the Protease Inhibitor Cocktail (Sigma-Aldrich). The lysates were resolved in sodium dodecyl sulfate polyacrylamide gels and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% fat-free milk and incubated with anti-PAX8, anti-E-cadherin, anti-vimentin, anti-vascular endothelial growth factor (VEGF), anti-FOXM1, anti-FOXC2, anti-FOXF1, anti-FOXL1, and anti-β-actin (Abcam, Cambridge, MA, USA) at 4 °C overnight. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h. Signals were developed by enhanced chemiluminescence (Merck Millipore, Darmstadt, Germany).
Anti foxm1
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-FOXM1 is a primary antibody that specifically recognizes the FOXM1 protein. FOXM1 is a transcription factor that plays a key role in cell cycle regulation and is involved in various cellular processes. The antibody can be used for applications such as Western blotting, immunohistochemistry, and immunofluorescence to detect and study the expression and localization of FOXM1 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (2 mentions)
Anti flag, by Merck Group (2 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Enhanced chemiluminescence, by Merck Group (1 mentions)
Anti e cadherin, by Abcam (1 mentions)
Anti vimentin, by Abcam (1 mentions)
Anti β actin, by Abcam (1 mentions)
Anti pax8, by Abcam (1 mentions)
Anti foxc2, by Abcam (1 mentions)
Anti foxf1, by Abcam (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Anti ubiquitin, by Abcam (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Bicinchoninic acid kit, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride membrane filter, by Merck Group (1 mentions)
Anti glyceraldehyde 3 phosphate dehydrogenase gapdh, by Abcam (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Fasc calibur, by BD (1 mentions)
Anti sox2, by Cell Signaling Technology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
12 protocols using anti foxm1
1
Western Blot Analysis of Cell Signaling
2
Regulation of FOXM1 Ubiquitination by OTUB1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Caki-1 and 786-O cells transfected with siOTUB1 or siNC were firstly treated with 10 μM MG132 for 3 h and then collected and lysed. The lysates were immunoprecipitated by anti-FOXM1 (Abcam) and immunoblotted by anti-ubiquitin (Abcam). For measurement of endogenous FOXM1 turnover rate, Caki-1 and 786-O cells transfected with siOTUB1 or siNC were treated with 80 μg/mL cycloheximide (CHX) (Sigma-Aldrich, St. Louis, MO, USA) for inhibition of protein synthesis. At 0, 1, 2, 4 h, cells were harvested and analyzed by western blot.
3
Western Blot Analysis of FOXM1 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein was extracted from tissue samples, and the concentration was determined using a bicinchoninic acid kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein (40 μg) were separated by 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membrane filters (Millipore, Billerica, MA, USA). Briefly, 5% non‐fat dry milk was used to block the non‐specific binding. Membranes were incubated overnight at 4°C with primary antibodies anti‐FOXM1 (1:500) and anti‐glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (1: 5000; Abcam, Cambridge, MA, USA). After washing, the membranes were incubated with corresponding secondary antibodies (horseradish peroxidase‐conjugated goat anti‐rabbit antibodies, 1: 5000; ZSGB Biotech, Beijing, China) for one hour at room temperature. Finally, the protein levels were quantified by an enhanced chemiluminescence (ECL) detection system (Amersham Imager 600; General Electric, Fairfield, CT, USA).
4
Analyzing GBM Cell Cycle and Stemness
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Patient-derived GBM cells were dissociated into single cells, and then permeabilized using the BD Cytofix/CytopermTM buffers (BD Biosciences, San Jose, CA, USA). The cells were labeled with the following antibodies: anti-FoxM1 (Abcam, UK), anti-Sox2 (Cell signaling, USA). To analyze the cell cycle, cells were fixed with 100% ethanol and incubated at 4 C for a day. The cell cycle disposition of cells stained with PI (Sigma Aldrich, USA) was analyzed using Flow Cytometry (FASC Calibur, BD Biosciences, San Jose, CA, USA), and the flow cytometry data were analyzed with a Flow JoTM software (Treestar Inc, USA)
5
Immunoprecipitation and Western Blotting Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole cell lysates were obtained by re-suspending cell pellets in RIPA buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% Triton X-100) with freshly added protease inhibitor (Roche) as previously described [37 ]. Nuclear proteins were prepared with the NE-PER Kit (Pierce) following manufacturer's recommendation. Specific antibodies or pre-immune IgGs (P.I.I.) were added to and incubated with cell lysates overnight before being absorbed by Protein A/G-plus Agarose beads (Santa Cruz). Precipitated immune complex was released by boiling with 1X SDS electrophoresis sample buffer. Alternatively, FLAG-conjugated beads (M2, Sigma) were added to and incubated with lysates overnight. Precipitated immune complex was eluted with 3X FLAG peptide (Sigma). Western blotting was performed with anti-FLAG (Sigma, F3165), anti-GFP (Proteintech, 50430-1), anti-MKL1 (Proteintech, 21166-1), anti-E2F1 (Cell Signaling Tech, 3742), anti-FOXM1 (Abcam, ab207298), anti-phosphorylated serine/threonine (Cell Signaling Tech, 9631), anti-MK2 (Cell Signaling Tech, 3042), anti-phosphorylated MK2 (Cell Signaling Tech, 3316), and anti-β-actin (Sigma, A2228).
6
Protein Expression Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Homogenization of the cells for 30 min in the lysis buffer on ice (50 mM Hepes, 5 mM EDTA, 150 mM NaCl, 50 mM NaF, 20 mM β‐glicerophosphate, 1 mM dithiothreitol [DTT] and protease inhibitor cocktail [Roche]) was done. Centrifugation of the lysates for 10 min at 4°C was done to clarify them. Proteins (25 μg/lane) were fractionated on 10% SDS‐polyacrylamide gels and transfer‐embedded onto nitrocellulose membranes. Rabbit polyclonal anti‐mTOR (CST), anti‐MYC (Abcam), anti‐CDK1 (Abcam), anti‐FOXM1 (Abcam), anti‐CCNB1 (Sigma), anti‐E2F6 (Abcam) and mouse monoclonal anti‐GAPDH (Santa‐Cruz) were used in immunoblots. Immunostained bands were developed and detected by the chemiluminescent method.
7
Western Blot Analysis of FOXM1 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After the protein was extracted, the concentration was detected by a BCA kit (Beyotime). After denaturation, the protein was separated on 12% SDS-PAGE gels and then transferred to PVDF membranes (Beyotime). After blocking with 5% nonfat milk, membranes were incubated with the following primary antibodies: anti-FOXM1 (1:1000, Abcam, UK) and anti-GAPDH (1:1000, Abcam). After PBST washes, the membranes were incubated with an HRP-conjugated anti-mouse IgG antibody (1:2000, Beyotime, China) at room temperature for 1 h. Finally, the bands on the membranes were visualized with the ECL plus reagent (Beyotime) and photographed by a Tanon-5200 automatic chemiluminescence imaging analysis system (Tanon, Shanghai, China).
8
Western Blot Analysis of Key Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analysis was performed as previously described [8 (link)]. Cells were lysed in RIPA lysis buffer. Cellular proteins were subjected to SDS-PAGE and western blot analysis with the following primary antibodies: anti-WW45 (Cell Signaling Technology), anti-Patched (Cell Signaling Technology), anti-FOXM1 (Abcam), anti-GAPDH (Santa Cruz), anti-Gli1 (Abcam), anti-ubiquitin (Cell Signaling Technology).
9
Western Blot and Immunoprecipitation Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot (WB) and immunoprecipitation /immunoblotting (IP/IB) analyses were performed on whole cell lysates and nuclear fractions according to published methods [23 , 24 (link)]. The anti-β-catenin, anti-PLK1, anti-phospho-PLK1 (Thr210), anti-AURKA, anti-phospho-AURKA (Thr288) and anti-GADD45α (D17E8) were purchased from Cell Signaling Technology. The anti-FOXM1 was purchased from Abcam. The anti-β-actin antibody used as loading control was purchased from Santa Cruz Biotechnology. Signal intensities in single blots obtained in three separate experiments were measured by means of ChemiDoc-It instrument (UVP) equipped with a dedicated software (Launch VisionWorksLS from Euroclone). The differences among signal intensities were evaluated for statistical significance using the paired Student’s t-test.
10
Protein expression analysis by Western blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The proteins were extracted and the concentrations were measured with a BCA protein assay Kit (Beyotime). Then Western blot assay was performed to detect the specific protein as previously described [48 (link)]. The target protein levels were normalized against that of the control α-tubulin. The primary antibodies anti-FOXM1 and anti-α-tubulin were purchased from Abcam corporation (Cambridge, MA, USA), and anti-cyclin B1 and anti-cyclin D1 were bought from Cell Signaling Technology (Boston, MA, USA). The second antibodies horseradish peroxidase-labeled goat anti-rabbit IgG and goat anti-mouse IgG were from Zhongshan Biotechnologies (Beijing, China). The ECL chemiluminescence kit was from Beyotime.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!