HEK293T cells, L cells (an immortalized mouse fibroblast cell line), SW480 cells and HCT116 cells were obtained from Shanghai Life Academy of Sciences cell library (Shanghai, China). All cells were grown in DMEM medium (Invitrogen, Carlsbad, CA), and maintained in culture supplemented with 10% heat-inactivated fetal calf serum, 100 ug ml−1 of penicillin, and 100 μg ml−1 of streptomycin at 37 °C with 5% CO2 in a humidified incubator (Thermo, Waltham, MA). During the study, all cell cultures were periodically tested for mycoplasma by using MycoAlert Mycoplasma Detection Kit (Lonza, Rockland, ME). For western blot, antibodies specific for VGLL4 (1:500, ab140290), TEAD4 (1:500, ab58310), Histone H3 (1:1,000, ab6002) and β-catenin (1:1,000, ab2365) were purchased from Abcam (Cambridge, UK); those for FLAG (1:5,000, M4439) and α-tubulin (1:2,000, T6199) were from Sigma (St. Louis, MO); and those for TCF4 (1:500, sc-166699), c-Jun (1:1,000, sc-4113) and GST (1:1,000, sc-138) were bought from Santa Cruz Biotechnology (Santa Cruz, CA).
Ab140290
Manufactured by Abcam
Sourced in United States
Ab140290 is a primary antibody that recognizes the Integrin beta 1 protein. It is suitable for use in various immunological applications such as immunohistochemistry and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Humidified incubator, by Thermo Fisher Scientific (1 mentions)
T6199, by Merck Group (1 mentions)
Ab2365, by Abcam (1 mentions)
Ab58310, by Abcam (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Mycoalert mycoplasma detection kit, by Lonza (1 mentions)
Ab6002, by Abcam (1 mentions)
M4439, by Merck Group (1 mentions)
Sc 166699, by Santa Cruz Biotechnology (1 mentions)
Sc 138, by Santa Cruz Biotechnology (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ecl reagent, by Merck Group (1 mentions)
Horseradish peroxidase conjugated igg secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Ab181602, by Abcam (1 mentions)
Pierce bca protein assay kit, by Tiangen Biotech (1 mentions)
Protein lysis buffer, by Beyotime (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Ab205270, by Abcam (1 mentions)
4 protocols using ab140290
1
Cell Culture and Antibody Reagents
2
Western Blot Analysis of VGLL4 Expression
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The transfected HCC cells were lysed by protein lysis buffer (Beyotime, Shanghai, China) and the concentrations of protein was measured using a Pierce BCA protein assay kit (Tiangen Biotech Co., Ltd., Beijing, China). A total of 20‐30 μg protein was separated by 10% SDS‐PAGE and electro‐transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). After being blocked with 5% skimmed milk, the membranes were incubated with VGLL4 (ab140290; Abcam, Cambridge, MA, USA) and GAPDH antibodies (ab181602; Abcam) overnight at 4°C. After incubation with the horseradish peroxidase‐conjugated IgG secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The protein expression levels were detected by ECL reagent (Millipore) and imaged using Amersham Imager 600 from GE Healthcare Life Sciences (Beijing, China). The blots were semi‐quantified by ImageJ software (1.46; National Institutes of Health, Bethesda, MD, USA).
3
Protein Extraction and Western Blot Analysis
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Proteins were extracted from the tissue samples according to the manufacturer’s instruction. Western blot analysis were conducted as previously described27 (link). The antibodies used in the study were shown as follow: anti-YAP (ab205270, Abcam, Cambridge, MA, USA), VGLL4 (ab140290, Abcam, Cambridge, MA, USA).
4
Evaluating VGLL4 Expression in Colon Cancer
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CRC and normal tissue microarray sections were prepared by Shanghai Outdo Biotech Co. Ltd. (Shanghai, China). This tissue array contains tissues from 40 human patients containing normal colons and 60 patients with CRC to examine the expression profiles of VGLL4 by IHC. For IHC, TMA sections were incubated with anti-VGLL4 antibody (1:50 dilution; ab140290; abcam). VGLL4 staining were scored by two independent pathologists, blinded to the clinical characteristics of the patients. The scoring system was based on the staining intensity and extent. Staining intensity was classified as 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). Staining extent was dependent on the percentage of positive cells (examined in 200 cells) were divided into 0 (<5%), 1 (5–25%), 2 (26–50%), 3 (51–75%) and 4 (>75%). According to the staining intensity and the staining extent scores, the IHC result was classified as 0–1, negative (−); 2–4, weakly positive (+); 5–8, moderately positive (++) and 9–12, strongly positive (+++).
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