Cells were lysed in lysis buffer (phosphate-buffered saline containing 1% Triton X-100, protease inhibitor cocktail, and 1 mM phenylmethylsulfonyl fluoride) at 4°C for 30 min. Lysate protein concentration was estimated using BCA protein assay kit (Pierce, USA). Equal amounts of protein were electrophoresed under non-reducing conditions on 10% acrylamide gels, followed by transfer to a PVDF membrane on a semidry transfer apparatus. After being blocked with 5% nonfat milk for 1 h at room temperature, the membrane was incubated with antibodies against FOXM1, MMP-2, MMP-9, and VEGF-A (ProteinTech Group, China) with proper dilutions for 1 h, respectively. After washing, horseradish peroxidase-conjugated anti-rabbit IgG (Sigma, USA) was used as a secondary antibody and then incubated with the membrane for 1 h at room temperature. The immunoreactive proteins were then detected using the ECL system. Densitometric analysis of immunoblots was performed by using Quantity One software.
Foxm1
Manufactured by Proteintech
Sourced in China
FOXM1 is a protein that plays a critical role in cell proliferation and cell cycle regulation. It functions as a transcription factor, activating the expression of genes involved in mitosis and cell division. The FOXM1 protein is commonly used in research related to cell biology, cancer biology, and developmental biology.
Automatically generated - may contain errors
Lab products found in correlation
Vegfa, by Proteintech (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Horseradish peroxidase conjugated anti rabbit igg, by Merck Group (1 mentions)
Cyclin a2, by Abcam (1 mentions)
Total stat3, by Cell Signaling Technology (1 mentions)
Phospho stat1 tyr701, by Cell Signaling Technology (1 mentions)
Phospho stat5 tyr694, by Cell Signaling Technology (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Phospho stat3 tyr705, by Cell Signaling Technology (1 mentions)
Rnf26, by Proteintech (1 mentions)
Gapdh, by Proteintech (1 mentions)
Protein a g beads, by Beyotime (1 mentions)
Easysee western blot kit, by Transgene (1 mentions)
Mybl2, by Proteintech (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
β actin, by Proteintech (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Cdc20, by Proteintech (1 mentions)
Protease inhibitor mixture, by Beyotime (1 mentions)
8 protocols using foxm1
1
Western Blot Analysis of Angiogenic Factors
2
Paraffin-Embedded Tissue IHC/IF Assay
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Tissues were fixed with 4% paraformaldehyde at 4 °C, while PDOs were fixed with 4% paraformaldehyde at room temperature for 15 min. Samples were embedded in paraffin. Then, the 5-μm-thick slices were subjected to IHC or IF assays. The antibodies used for staining were: ER (Proteintech, 21244–1-AP), PR (Proteintech, 25871–1-AP), and Ki67 (Proteintech, 27309–1-AP), FOXM1 (Proteintech, 13147–1-AP).
3
Protein Extraction and Western Blot Analysis
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Liver samples were homogenized in lysis buffer containing 100 mM Tris, pH 8.5, 250 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, aprotinin at 1:5000 dilution, and leupeptin at 1:5000 dilution46 (link). Tissue homogenates were centrifuged and the supernatants including tissue protein extracts were boiled in Laemmli buffer containing 10 mM dithiothreitol, then subjected to SDS-polyacrylamide gel electrophoresis. Separated proteins were transferred to nitrocellulose membranes and blocked in Tris-buffered saline containing 3% FBS. Immunoblot analyses were performed using antibodies to phospho-STAT3 (Tyr705) (#9131, Cell Signaling Technology, Danvers, MA, USA), total STAT3 (#4904, Cell Signaling Technology), phospho-STAT1 (Tyr701) (#9167, Cell Signaling Technology), phospho-STAT5 (Tyr694) (#9314, Cell Signaling Technology), FoxM1 (13147-1-AP, Proteintech, Rosemont, IL, USA), Cyclin A2 (ab181591, Abcam, Cambridge, UK), Cdk1 (#28439, Cell Signaling Technology), and PLK1 (#4535, Cell Signaling Technology) at 1:2000 dilution and actin (A2066, Sigma) at 1:5000 dilution. Quantitative data were obtained employing a ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Hercules, CA, USA). Uncropped membrane images are presented in Supplementary Fig. 6 .
4
Co-IP and Western Blotting for Bladder Cancer
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For co-IP, cells harvested from 10-cm plates were lysed in RIPA buffer (#P0013C, Beyotime, China) on ice for 30 min. The supernatant was collected after centrifugation (12,000 × g, 15 min) and cocultured with protein A + G beads (#P2029, Beyotime, China) and IgG antibody or primary antibody at 4 °C overnight. The next day, the beads were washed with RIPA buffer six times. The protocols for the use of human tissue (12 pairs of matched bladder cancer/adjacent noncancerous tissues) were approved by the local ethics committee (the Second Xiangya Hospital, Central South University). For western blotting, the cells were harvested and lysed in RIPA buffer on ice for 30 min. The supernatant was collected after centrifugation (12,000 × g, 15 min). Then, 4× loading buffer was added to the supernatant and boiled in hot water (100 °C) for 10 min. The supernatant was then subjected to electrophoresis on SDS-PAGE gels. The detailed protocol was reported previously [12 (link)]. The primary antibodies used were as follows: GAPDH (Proteintech, #60004-1-Ig, 1:5000 dilution), RNF26 (Proteintech, #16802-1-AP, 1:800 dilution), p57 (Proteintech, #23317-1-AP, 1:800 dilution), p53 (Proteintech, #10442-1-AP, 1:3000 dilution), and FOXM1 (Proteintech, #13147-1-AP, 1:1000 dilution). ImageJ software (National Institutes of Health) was used to evaluate the protein levels.
5
Western Blot Analysis of Protein Expression
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Protein was lysed from cells by using RIPA buffer (Cat no. 89901; Thermo Fisher, MA, USA) followed by centrifugation at 16,000 × g and 4°C for 15 min. To quantify the protein concentration, a bicinchoninic acid (BCA) protein assay (Cat no. 23225; Thermo Fisher, MA, USA) was performed. Denaturation of total protein was processed in a metal bath at 95°C for 5 min, and then, 30 μg of total proteins was separated by SDS-PAGE (7.5%). After that, proteins were transferred to PVDF membranes. 5% skimmed milk was dissolved in TBST to block the PVDF membrane for 2 h at room temperature. Then, the membranes were washed and incubated with the primary antibodies overnight.
The primary antibodies included anti-MTDH (1 : 1500) (Cat no. 13860-1-AP; Proteintech, Wuhan, Hubei, China), MYBL2 (1 : 1500) (Cat no. 18896-1-AP; Proteintech, Wuhan, Hubei, China), FoxM1 (1 : 1500) (Cat no. 13147-1-AP; Proteintech, Wuhan, Hubei, China), and β-actin (1 : 2000) (Cat no. CL488-66009; Proteintech, Wuhan, Hubei, China) antibodies. Then, the membranes were incubated with the horseradish peroxidase- (HRP-) conjugated secondary antibody (1 : 1000) (Cat. no. 7074; Cell Signaling Technology, Inc.) for 2 h at 37°C. To measure the chemiluminescence signals, the EasySee Western Blot Kit (Beijing TransGen Biotech, Beijing, China) was utilized. ImageJ version 1.53 software was used to quantify the proteins.
The primary antibodies included anti-MTDH (1 : 1500) (Cat no. 13860-1-AP; Proteintech, Wuhan, Hubei, China), MYBL2 (1 : 1500) (Cat no. 18896-1-AP; Proteintech, Wuhan, Hubei, China), FoxM1 (1 : 1500) (Cat no. 13147-1-AP; Proteintech, Wuhan, Hubei, China), and β-actin (1 : 2000) (Cat no. CL488-66009; Proteintech, Wuhan, Hubei, China) antibodies. Then, the membranes were incubated with the horseradish peroxidase- (HRP-) conjugated secondary antibody (1 : 1000) (Cat. no. 7074; Cell Signaling Technology, Inc.) for 2 h at 37°C. To measure the chemiluminescence signals, the EasySee Western Blot Kit (Beijing TransGen Biotech, Beijing, China) was utilized. ImageJ version 1.53 software was used to quantify the proteins.
6
Investigating Cell Cycle Regulators
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Cells were prepared using RIPA buffer (Beyotime) containing protease inhibitor mixture (Beyotime). Immnoprecipitates or total cell lysates were analyzed by western blot according to standard procedures. The antibodies used in this study were against: Cdc20 (Proteintech, 10252-1-AP, 1:1000), Cdh1 (Santa, sc-56312 1:1000), FoxM1 (Proteintech, 13147-1-AP, 1:1000), Cyclin B1 (CST, 4138T, 1:1000), Cyclin B2 (Beyotime, AF2509, 1:1000), Apc (Beyotime, AF2113, 1:1000), β-catenin (Proteintech, 51067-2-AP, 1:1000), HA (Proteintech, 66006-2-Ig, 1:10,000), and Tubulin (Beyotime, AF0001, 1:1000).
7
Western Blot Analysis of FoxM1 and VEGF-A
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Western blot analysis was performed as previously reported. Briefly, the membranes were probed with the following primary antibodies: FoxM1 (1:1000, Proteintech, Wuhan, China), VEGF-A (1:1000, Proteintech, Wuhan, China) and β-actin (1:3000, Abways, Shanghai, China). After being washed with TBS-T, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody (1:10000, Proteintech, Wuhan, China). Mouse anti-β-actin (1:1000, Proteintech, Wuhan, China) with goat anti-mouse (1:10000, Proteintech, Wuhan, China) antibodies were used as a loading control. Chemiluminescence detection was performed with ChemiGlow detection reagents (Bio-Rad Western ECL Substrate). The blots were visualized with a Bio-Rad ChemiDoc MP and quantified with a densitometer using the imager application program (Alpha Innotech, San Leandro, CA, United States).
8
Immunofluorescence Assay of Cell Markers
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Cells grown on slides were xed in 4% paraformaldehyde for 15 min, and then washed 3 times in PBS for 5 min each. The cells were permeabilized by treatment with 0.5% triton for 10 min, and then washed 3 times with PBS for 5 min each. Cells were blocked for 30 min using undiluted goat serum. Next, cells were incubated with primary antibody (0.5 mg /mL BSA dilution, 1: 200) overnight at 4 ℃. After rinsed 3 times with PBST, the secondary antibody (0.5 mg / mL BSA dilution, 1: 200) was added and incubated at room temperature for 2h in the dark. After 3 times washed with PBST, nuclei were stained for 10 min, then rinsed with PBS 3 times. Then the slides were observed using uorescence microscope. Antibodies used in immuno uorescence including FOXM1, Vimentin, E-cadherin, Snail and Cy3-linked anti-rabbit were purchased from Proteintech.
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