Protein lysates were prepared with RIPA Buffer (PI-89900, Thermo Fisher Scientific) and quantified using BCA Protein Assay Kit (23225, Pierce Biotechnology). Equal amounts of total protein were loaded for immunoblot analysis. Immunoblots were probed with antibodies against MYC (5605S, Cell Signaling Technology), SMAD4 (38454S, Cell Signaling Technology), GLI3 (sc-74478, Santa Cruz Biotechnology), MYBL2 (sc-390198, Santa Cruz Biotechnology), LOXL2 (ab96233, Abcam), CLDN1 (4933S, Cell Signaling Technology), HGK (3485, Cell Signaling Technology), NRAS (ab77392, Abcam), E-cadherin (ab40772, Abcam), Vimentin (D21H3, Cell Signaling), LAMB1 (D4Q4Z, Cell Signaling) and GAPDH (MA5–15738, Thermo Fisher Scientific) overnight at 4°C followed by incubation for 1 hr at room temperature with corresponding HRP conjugated goat anti-mouse (31430, Thermo Fisher Scientific) or goat anti-rabbit (31460, Thermo Fisher Scientific) secondary antibodies. Western Blots were then developed using ECL detection kit (34096, Thermo Fisher Scientific) and captured on an Amersham Imager 600 (GE Healthcare). Densitometry analysis was performed using Image J software to quantify each protein band, and were normalized against LAMB1 for nuclear fraction or GAPDH.
Mybl2
Manufactured by Santa Cruz Biotechnology
MYBL2 is a protein-coding gene that provides instructions for making a protein involved in the regulation of cell division. This protein plays a role in the process of cell proliferation and is expressed in various tissues and cell types.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Cldn1, by Cell Signaling Technology (1 mentions)
Smad4, by Cell Signaling Technology (1 mentions)
Ecl detection kit, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
E cadherin, by Abcam (1 mentions)
Non specific immunoglobulin igg, by Merck Group (1 mentions)
Foxm1, by Santa Cruz Biotechnology (1 mentions)
Imagequant las 4000 imager, by GE Healthcare (1 mentions)
Brca2, by Cell Signaling Technology (1 mentions)
Brca1, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
3 protocols using mybl2
1
Western Blot Characterization of Protein Markers
2
Immunohistochemical Analysis of MYBL2 and FoxM1
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TMA slides were processed and stained manually as described previously. Formalin- fixed, paraffin-embedded sections were prepared for all tissues. Sections were deparaffinized in xylene and rehydrated through graded alcohol to water, and endogenous peroxidase activity was blocked by incubating the slides in 3% H2O2 in water for 30 min at room temperature. Sections were incubated in 1% BSA for 30 min then wiped off and dilution of MYBL2 (1:200, Santa Cruz) and FoxM1 (1:200, Santa Cruz) were applied to the slides and incubated overnight at room temperature. Subsequently, sections were incubated with secondary antibody for 2 h at room temperature, according to the manufacturer’s instructions. Negative control slides were processed in parallel using a non-specific immunoglobulin IgG (Sigma Chemical Co, St. Louis, MO, USA) at the same concentration as the primary antibody. Stained sections were observed under a microscope. Only fresh cut slides were stained simultaneously to minimize the influence of slide ageing and maximize repeatability and reproducibility of the experiment.
3
Western Blot Analysis of DNA Repair Proteins
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Protein lysates were prepared by homogenizing the tumor tissues in 4% SDS. Protein concentration was determined using Pierce BCA protein assay kit (Thermo Scientific #23225). Equal amount of protein (~40 µg) was loaded and resolved on a 10% or 12% SDS polyacrylamide gel and then transferred to PVDF membranes. The membranes were probed with antibodies against BRCA1 (Cell Signaling Technology #9010, 1:1000 dilution), BRCA2 (Cell Signaling Technology #10741, 1:1000 dilution), RAD51 (Abcam ab133534, 1:1000 dilution), FANCD2 (Abcam ab108928, 1:1000 dilution), FANCA (Proteintech 11975-1-AP, 1:1000 dilution), MYBL2 (Santa Cruz Biotechnology sc-81192, 1:1000 dilution), cleaved Caspase-3 (Cell Signaling Technology #9661, 1:500 dilution) and PARP (BD Pharmingen 556494, 1:500 dilution). Actin (Abcam ab3280, 1:3000 dilution) or GAPDH (Abcam ab8245, 1:2000 dilution) antibodies were used as protein loading controls. HRP conjugated anti-rabbit IgG (Bio-Rad 1706515) or anti-mouse IgG (Bio-Rad 1721011) secondary antibodies were used at 1:10000 dilution. Blots were visualized on ImageQuant LAS 4000 imager (GE Healthcare Life Sciences).
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