Total protein was extracted using lysis buffer (ThermoFisher, Rockford, IL) and Bradford method was used to quantify the protein. When SDS-PAGE assay was performed, 30 g of the protein was separated and then transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). For primary antibody incubation, cleaved caspase 3, cleaved YAP, Bcl-2, GLUT-3 (1:1000; Cell Signaling Technology, Danvers, MA, USA), and GAPDH (1:2000; Cell Signaling Technology, Danvers, MA, USA) were incubated overnight at 4°C. For secondary antibody incubation, peroxidase-coupled anti-mouse/rabbit IgG (1:1000, Cell Signaling Technology, USA) was incubated at 37°C for 2 hrs. The result was visualized using ECL (Thermo, Rockford, IL) and detected using a DNR BioImaging System (DNR, Jerusalem, Israel). The quantification of WB band density was performed by using Photoshop CS6 (Adobe).
Glut3
Manufactured by Cell Signaling Technology
Sourced in United States
GLUT3 is a protein that functions as a glucose transporter. It facilitates the diffusion of glucose across cell membranes.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Lysis buffer, by Thermo Fisher Scientific (1 mentions)
Peroxidase coupled anti mouse rabbit igg, by Cell Signaling Technology (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Amersham enhanced chemiluminescence, by GE Healthcare (1 mentions)
Notch1, by Cell Signaling Technology (1 mentions)
Glut1, by Cell Signaling Technology (1 mentions)
Tead1, by Santa Cruz Biotechnology (1 mentions)
Jagged1, by Santa Cruz Biotechnology (1 mentions)
Pepck, by Cell Signaling Technology (1 mentions)
Aldoa, by Cell Signaling Technology (1 mentions)
Cell extraction buffer, by Thermo Fisher Scientific (1 mentions)
Anti mouse or anti rabbit igg, by GE Healthcare (1 mentions)
P taz ser89, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
5 protocols using glut3
1
Quantitative Protein Analysis and Western Blot
2
Western Blot Analysis of Cellular Proteins
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Total protein was isolated from cells using Cell Extraction Buffer (Biosource, Camarillo, CA) supplemented with protease and phosphatase inhibitors and precleared using centrifugation, followed by measuring protein concentrations using the BCA Protein Assay kit (Pierce, Rockford, IL). The primary antibodies including TAZ, CTGF, Notch1, Hes1, LDHA, PFKB3, PEPCK, PKM2, PGK1, HK2, GLUT1, GLUT3, ALDOA, p-TAZ (Ser89), and β-actin were purchased from Cell Signaling Technology (CA, US). Jagged1 and TEAD1 were purchased from Santa Cruz Biotechnology (CA, US). Appropriate secondary antibodies conjugated to horseradish peroxidase were used, including anti-mouse or anti-rabbit IgG (GE-Healthcare, CA, US). Proteins were visualized by Amersham enhanced chemiluminescence (GE-Healthcare, CA, US). Immunohistochemistry assay was performed as previously described [7 (link)].
3
Western Blot Protein Analysis Protocol
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Total proteins were harvested and extracted using lysis mixture with phosphatase inhibitor cocktail and protease inhibitor cocktail. Then, the protein samples were quantified and the same amount of proteins were transferred to PVDF membranes (EMD Millipore, MA, USA). The membranes were blocked with BSA at room temperature for 2 hours. Then, the membranes were washed with TTBS buffer and incubated with primary antibodies at 4°C overnight. Primary antibodies used were listed as follows: TRIM66 (1:1000, Sigma, USA), GLUT3 (1:800, Cell signaling technology, USA), cMyc (1:3000, Abcam, UK). Then, membranes were incubated with HRP-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies for 2 h at 37°C. The immunoreactive bands were detected and reported using SuperSignal West Dura Extended Duration Substrate (Thermo, MA, USA) and Bio-Imaging Systems (DNR, Israel).
4
Western Blot Analysis of Protein Markers
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For Western blot, 60ug protein was separated by SDS-PAGE. Then proteins were transferred to PVDF membrane and incubated with primary antibody including Six1 (1:800; Sigma, USA), MMP2, GLUT3, Snail (1:1000; Cell Signaling Technology, USA), and GAPDH (1:2000; Santa Cruz, USA). Membranes were washed with TBS-T, and incubated with HRP-conjugated secondary antibody (1:2000, Santa Cruz, USA) for 1 hour. Images were taken using the DNR Imaging Systems after ECL development (ThermoFisher, USA).
5
Glioblastoma Immunohistochemical Analysis
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Human Brain Cancer Glioblastoma (grade IV) and normal brain samples were purchased from US Biomax, Inc., and mouse brain tissues was prepared in the Laboratory of Animal Research at the Asan Medical Center. The tissues were sectioned 4 μm thick on paraffin-embedded slides. Tissue slides were incubated at 60 °C for 1 h and then deparaffinized with xylenes and rehydrated with 100, 95, 90, 85, 50, and 0% ethanol. The primary antibody was incubated in the tissues overnight at 4 °C. Antibodies used include HDAC2 (Santa cruz, 1:1000), Bax (Cell signaling, 1:1000), Apaf-1 (Cell signaling, 1:1000), and GLUT3 (Cell signaling, 1:1000). The IHC process was carried out using the PROCAM IHC kit (Abcam, MA USA). Digital images were obtained through (OLYMPUS-cellSens Standard). Quantitative analysis of the images was performed using Image J (NIH).
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