MDA-MB-231 cells were seeded into 6-well plates and treated for 5 days with either 0 or 100 μM vitamin C. Cell lysates were collected in radioimmunoprecipitation assay (RIPA) buffer (Thermo Scientific, Waltham, MA, USA) containing a protease inhibiter cocktail (Sigma-Aldrich, St. Louis, MO, USA), 1% SDS, and 0.5 mM dithiothreitol (DTT). The protein concentration was determined by the BCA protein assay (Thermo Scientific, Waltham, MA, USA). Cell lysates were resolved by SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories, Hercules, CA, USA), and immunoblotted with anti-HIF-1α antibody, anti-YAP1 antibody, anti-cyclophilin B (PPIB) antibody and, anti-β-actin antibody (Cell Signaling Technology, Danvers, MA, USA). Proteins were visualized by using chemiluminescence ECL (Thermo Scientific, Waltham, MA, USA). Specific band densities were quantified by using ImageJ and analyzed by Student’s t-test, at α = 0.05.
Anti yap1 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-YAP1 antibody is a tool used to detect and study the transcriptional co-activator YAP1 protein in biological samples. It is a highly specific and well-characterized reagent for the identification and analysis of YAP1 expression and localization.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Protease inhibiter cocktail, by Merck Group (1 mentions)
Anti hif 1α antibody, by Cell Signaling Technology (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Anti β actin antibody, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride pvdf membrane, by Bio-Rad (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti rad51 antibody, by Proteintech (1 mentions)
Pierce immunoprecipitation kit, by Thermo Fisher Scientific (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Ultravision detection system, by Lab Vision (1 mentions)
Phalloidin ifluor 594, by Abcam (1 mentions)
6 protocols using anti yap1 antibody
1
Vitamin C Regulates HIF-1α and YAP1 in MDA-MB-231 Cells
2
Immunohistochemical Analysis of YAP1 Expression
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Paraffin-embedded samples were first dewaxed by xylene followed by immersing in 100% ethanol for three times (5 minutes/time). Later, slides were sequentially immersed in 95%, 80%, and 70% ethanol and finally washed by double distilled water. Then, de-waxed samples were subjected to antigen retrieval by immersing in citrate buffer (pH 6.0) followed by a treatment with 3% H2O2 for 5 min. Next, samples were hybridized with antibody in a humidity chamber at 4 °C overnight. After washing for 1 h, color was developed by an AEC substrate buffer (Bio SB, Inc., Santa Barbara, CA, USA) followed by hematoxylin staining (Bio SB, Inc., Santa Barbara, CA, USA). Finally, samples were mounted with gelatin.
Following treatments, cells were fixed for 10 min with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 in PBS washing buffer for 30 min, and blocked with 5% normal goat serum for 1 h at room temperature. Cells were then stained with anti-YAP1 antibody (Cell Signaling #14074, 1:100, Danvers, MA, USA) at 4 °C overnight. After washing, cells were treated with Goat Anti-Rabbit IgG Alexa Fluor 488 conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA USA) for 1 h and counterstained with DAPI for nuclei.
Following treatments, cells were fixed for 10 min with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 in PBS washing buffer for 30 min, and blocked with 5% normal goat serum for 1 h at room temperature. Cells were then stained with anti-YAP1 antibody (Cell Signaling #14074, 1:100, Danvers, MA, USA) at 4 °C overnight. After washing, cells were treated with Goat Anti-Rabbit IgG Alexa Fluor 488 conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA USA) for 1 h and counterstained with DAPI for nuclei.
3
Immunohistochemical Analysis of Tumor Markers
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IHC was performed with a normal inverted microscope (NION, Japan) by using an anti-YAP1 antibody (1:200; Cell Signaling Technology), anti-CPNE3 antibody (1:50; abcam, Proteintech), anti-cysteine-rich angiogenic inducer 61 (CYR61) antibody (1:100; Proteintech), and anti-RAD51 antibody (1:100; Proteintech). IHC was performed as described previously [11 (link)]. Two pathologists assessed the staining results based on the proportion of positively stained cells and the staining strength.
4
Co-immunoprecipitation Assay for YAP1
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The co-IP assay was performed using the Pierce® immunoprecipitation kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, AGS cells were lysed by ice-cold IP lysis/wash buffer and centrifuged at 13,000×g for 10 min to remove the debris. The supernatant was further immunoprecipitated with an anti-YAP1 antibody (1:50; Cell Signaling Technology). Rabbit IgG was used as a negative control. Precipitates were separated using SDS-PAGE and further analyzed by performing immunoblotting.
5
Western Blotting and Immunohistochemistry Analysis
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For western blotting, 40 ug of protein lysates were used as described [57 (link)]. Antibodies used and conditions are listed in Supplemental Table 4 . Protein expression was analyzed using Odyssey infrared imaging system (Li-Cor) and the data were normalized on siOT and on shOT. Immunohistochemistry was performed using anti-YAP1 antibody diluted 1:25 (Cell Signaling, 4912) using Ultra vision detection system (LabVision), upon heat-induced epitope retrieval. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide in methanol for 30 minutes. Intensity scoring of tumor cells was performed by pathologist based on a 4-tiered scale. The comparisons between two classes were performed by Fisher two tailed extract test by grouping negative/1+ cases and 2+/3+ cases.
6
Visualizing YAP1 and F-actin in CRC Cells
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HCT116 WT or GPR4-depleted CRC were plated into 8-well u-Chamber (Ibidi, Germany) and incubated overnight. After that, the cells were fixed and permeabilized with buffer supplied with 0.05%Triton X-100 for 3 min; then, the cells were washed with PBS containing 0.05% Tween-20 twice. Next, the cells were blocked with 5% BSA buffer for 2 h at room temperature. For YAP1 staining, cells were incubated with anti-YAP1 antibody (#14074, 1:100, Cell signalling) overnight at 4 °C. On the second day, cells were washed twice and incubated with goat anti-rabbit antibodies conjugated with Alexa 488 at a 1:300 for 1 h. Cells were incubated with Phalloidin-iFluor 594 (ab176757, Abcam) to stain F-actin. After that, the cells were washed twice and supplied with anti-fade fluorescence medium with DAPI (S2110, Solarbio), and visualized with a confocal microscope.
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