Human glioma cell lines LN229 and U251 were obtained from the American Type Culture Collection (ATCC). LN229 and U251 cells were cultured with DMEM supplemented with 10% foetal bovine serum FBS (ExCell Bio, Lot: FSP500), 2 mM L-glutamine, penicillin (100 U/mL), streptomycin (100 μg/mL) and 0.1% Savelt™ (Hanbio Co. Ltd., 1:1000) in a humidified atmosphere of 5% CO2 maintained at 37 °C. The medium was replaced every day, and the cells were passaged before reaching confluence. The following antibodies were used in this study: Nanog (Cell Signaling Technology; #3580), OCT4 (Cell Signaling Technology; #2750), KLF4 (Santa Cruz Biotechnology, SC-20691), KLF4 (Cell Signaling Technology, #12173S), ITGB4 (Proteintech, 21738–1-AP), ITGB4 (Abcam, ab29042), VHL (Cell Signaling; #68547), Beta-actin (Proteintech, 6609–1-lg).
Itgb4
Manufactured by Proteintech
Sourced in United States
ITGB4 is a protein-coding gene that provides instructions for producing the beta 4 subunit of integrin, a cell surface receptor. Integrins play a role in mediating cellular adhesion and signaling. The ITGB4 gene product is a structural component of hemidesmosomes, which are adhesive complexes that connect the cytoskeleton of epithelial cells to the underlying basement membrane.
Automatically generated - may contain errors
Lab products found in correlation
Lumiglo peroxidase chemiluminescent substrate kit, by LGC (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Icam 1, by Proteintech (2 mentions)
Itgb4, by Abcam (1 mentions)
β actin, by Proteintech (1 mentions)
Nanog, by Cell Signaling Technology (1 mentions)
Foetal bovine serum (fbs), by ExCell Bio (1 mentions)
Ln229, by American Type Culture Collection (1 mentions)
Immun blot polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Cotl1, by Proteintech (1 mentions)
Fibronectin, by Sino Biological (1 mentions)
E cadherin, by Proteintech (1 mentions)
Vimentin, by Proteintech (1 mentions)
Goat anti mouse igg hrp, by Proteintech (1 mentions)
Gapdh, by Proteintech (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Hrp goat anti rabbit igg, by Proteintech (1 mentions)
Confocal microscope, by Zeiss (1 mentions)
Cy5 affinipure donkey anti goat igg, by Jackson ImmunoResearch (1 mentions)
Cy3 labeled donkey anti rabbit igg, by BioLegend (1 mentions)
5 protocols using itgb4
1
Characterization of Glioma Cell Lines
2
Western Blot Protocol for Protein Analysis
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Western blots were generated according to a previously described protocol [28 (link)]. Briefly, 5 µL protein lysates were loaded onto SDS-polyacrylamide gels and transferred to Immun-Blot polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA). After blocking in 5% milk, the membranes were incubated first in primary antibodies against p63 (4A4, 1:20,000), COTL1 (Proteintech, 1:10,000), K14 (a gift from Dr. Rose-Anne Romano) [29 (link)], Vimentin (CST, 1:5000), MMP9 (Proteintech, 1:10,000), Fibronectin (SinoBiological, 1:5000), ITGB4 (Proteintech, 1:10,000), E-cadherin (CST, 1:5000), and K6 (a gift from Dr. Julie Segre), then with horseradish peroxidase-conjugated secondary antibodies corresponding to the host of the primary antibody, and then washed in Tris-buffered saline with 0.05% Tween-20. Protein expression was detected with the LumiGLO peroxidase chemiluminescent substrate kit (SeraCare, Milford, MA, USA), and membranes were imaged using a Bio-Rad ChemiDoc imaging system. Uncropped Western blot images can be found in Figures S7–S9 .
3
Protein Extraction and Western Blot Analysis
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Protein from cells was extracted with RIPA buffer (Solarbio, Beijing, China). The sample was loaded onto 12% SDS-PAGE gel for electrophoresis and then transferred onto a PVDF membrane. After blocking by 5% skim milk at room temperature for 2 h, the membrane was incubated with primary antibody targeting NPR1 (1:2000, Thermo Fisher Scientific, Waltham, MA, USA), ITGB4 (1:1000, Proteintech, Wuhan, Hubei, China), ICAM-1 (1:1000, Proteintech, Wuhan, Hubei, China), and GAPDH (1:10,000, Proteintech, Wuhan, Hubei, China) at 4 °C overnight. Afterwards, incubation of secondary antibody HRP goat anti-rabbit IgG (1:10,000, Proteintech, Wuhan, Hubei, China) or HRP goat anti-mouse IgG (1:10,000, Proteintech, Wuhan, Hubei, China) was carried out at room temperature for 2 h. The protein band was detected by Tanon 5200 Chemiluminscent and Fluorescent Imaging System (Tanon, Shanghai, China).
4
Immunofluorescence Analysis of Vascular Markers
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Frozen sections were fixed with 4% neutral buffered paraformaldehyde (Bmassay, Beijing, China) for 10 min and then blocked with PBST containing 1% BSA for 2 h at room temperature. After blocking, the sections were incubated with primary antibody including NPR1 (1:100, Thermo Fisher Scientific, Waltham, MA, USA), ITGB4 (1:400, Proteintech, Wuhan, Hubei, China), ICAM-1 (1:200, Proteintech, Wuhan, Hubei, China), and CD31 (1:100, R&D, Minneapolis, MN, USA) at 4 °C overnight. Subsequently, the sections were incubated with secondary antibody Cy3-labeled donkey anti-rabbit IgG (1:200, Biolegend. San Diego, CA, USA) or Cy5-affinipure donkey anti-goat IgG (1:200, Jackson ImmunoResearch, West Grove, PA, USA) at room temperature 1 h. The nucleus was stained with Hochest3342 (Beyotime, Shanghai, China). The sections were examined by confocal microscope (Carl Zeiss, Oberkochen, Germany).
5
Western Blotting for Protein Expression
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Protein extracts were prepared according to previously published protocol (27 (link)). Briefly 5 μL of protein lysates were loaded onto SDS-polyacrylamide gels and transferred to Immun-Blot PVDF membranes (Bio-Rad Laboratories). After blocking in 5% milk, the membranes were incubated in primary antibodies against the following: p63 (4A4, 1:20,000), ΔNp63 (E6Q3O; Cell Signaling Technology, 1:5000), ITGB1 (Proteintech, 1:10,000), ITGB4 (Proteintech, 1:10,000), cMYC (Santa Cruz Biotechnology, 1:5000), AKT1 (Proteintech, 1:10,000), mTOR (Proteintech, 1:10,000), Raptor (Proteintech, 1:10,000), S6 (Cell Signaling Technology, 1:5000), and p-S6 (Cell Signaling Technology, 1:5000). The MAB374 antibody (EMD Millipore) was used to detect GAPDH as a loading control at 1:20,000 dilution. HRP-conjugated secondary antibodies corresponding to the primary antibody host were incubated with each blot. Unbound antibodies were washed off in 0.05% Tween-20 in Tris-buffered saline. The LumiGLO peroxidase chemiluminescent substrate kit (SeraCare) was used to detect antibody-labeled proteins, and membranes were imaged using the Bio-Rad ChemiDoc imaging system.
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