Immunoblotting was exercised to examine the expression of Hh signaling pathway-related markers in HCC cells according to standard protocols as described previously.18 (link) Protein was harvested using radio immunoprecipitation assay lysis buffer, and a bicinchoninic acid assay was applied to determine the protein concentration. Next, we separated equal amount of protein using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred the protein to a polyvinylidene difluoride membrane. After blocking with 1.5% skimmed milk for 1 h at room temperature, membranes were incubated with the primary antibodies at 4°C overnight and with horseradish peroxidase-conjugated secondary antibodies. Membranes were developed using a chemiluminescent substrate. The antibodies were used as follows: Gli1 (1:200, ab49314, Abcam), Gli2 antibodies (1:500, ab26056) and Gli3 antibodies (1:500, ab6050) and the secondary antibodies (1:50,000, ab7090).
Ab49314
Manufactured by Abcam
Sourced in United States, China
Ab49314 is a lab equipment product offered by Abcam. It is designed for specific laboratory applications, but a detailed description cannot be provided while maintaining an unbiased and factual approach. Further information on the core function and intended use of this product is not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab53715, by Abcam (5 mentions)
Ab53281, by Abcam (4 mentions)
Ab8245, by Abcam (3 mentions)
Ab32572, by Abcam (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ab26056, by Abcam (2 mentions)
Ab6050, by Abcam (2 mentions)
Ab7090, by Abcam (2 mentions)
Ab40772, by Abcam (2 mentions)
Ab167389, by Abcam (2 mentions)
Ab76057, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ab5694, by Abcam (2 mentions)
Sc 8008, by Santa Cruz Biotechnology (2 mentions)
Gant61, by Merck Group (2 mentions)
Annexin 5 fitc kit, by Abcam (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Ab38686, by Abcam (2 mentions)
Ab39253, by Abcam (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
26 protocols using ab49314
1
Immunoblotting Analysis of Hedgehog Signaling in HCC
2
Hedgehog Pathway Protein Expression
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After treatments, tumor cells were washed twice with PBS and then lysed. Protein concentration of each sample was determined by Bradford assay. Cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane, which was activated by methyl alcohol for five minutes before. The membrane was blocked for 1–2 h at room temperature with 5% non-fat milk, then washed with TBST buffer for three times and incubated overnight at 4 oC with one of the following antibodies: Anti-Gli1 (Abcam ab49314, 1:1000), Anti-Patched (Abcam ab39266, 1:1000), Anti-Smoothened (Bioss bs-2801R) and anti-tubulin (Beyotime AT819, 1:1000). Chemiluminescence detection was performed with the corresponding second antibody conjugated with HRP. Images were acquired using BeyoECL plus and CLiNX Science instruments.
3
Protein Quantification and Western Blot Analysis
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The D17 cell line was treated with 20 µM GANT61 or vehicle DMSO for 96 hrs and cell lysate was made in lysis buffer. Protein was quantified using a BCA protein Assay Reagent (bicinchonic acid) (Pierce). A total of 40 µg of protein was loaded per sample in the 7.5% polyacrylamide gels under denaturing and reducing conditions and protein was transferred to nitrocellulose membranes. After transfer of protein, the membrane was probed and incubated overnight at 4°C with antibodies Rabbit anti-GLI1 (Abcam ab49314), Rabbit anti-GLI2 (Abcam ab26056), Rabbit anti-Patch1(Sigma-Aldrich P0088), Rabbit anti-Pax6 (Abcam ab5790), and Mouse anti-Actin (Santa Cruz sc-56459) in 5% non-fat milk. Then membranes were washed and subsequently exposed to the appropriate HRP-conjugated secondary antibodies for 1 hr. at room temperature. Bands were visualized by the enhanced chemiluminescence (Pierce) in Fluorchem E Imaging system (Protein Simple, CA). Constitutive expression levels of GLI1 and GLI2 were performed similarly, in the absence of treatment for Abrams, D17 and Moresco cell lines.
4
Protein Expression Analysis in Cancer Cells
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LoVo or SW1116 cells were lysed on ice with the lysis buffer (Beyotime, Shanghai, China) and the lysates were later centrifuged for 15 min at 12,000 × g. The BCA kit (Thermo Fisher Scientific, Waltham, MA, USA) was acquired for evaluating protein concentrations. Protein was subjected to SDS–PAGE (Bio-Rad, Hercules, CA, USA) and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Primary antibodies and secondary antibodies were applied to incubate membranes in sequence. ECL detection system (Applied Biosystems, Foster City, CA, USA) was utilized to visualize protein bands. Primary antibodies against MMP2 (ab97779, Abcam, Cambridge, MA, USA), MMP7 (ab5706, Abcam), N-cadherin (ab76057, Abcam), E-cadherin (ab40772, Abcam), NANOG (ab80892, Abcam), OCT4 (ab181557, Abcam), Gli4 (AV37797, Sigma-Aldrich), PTCH1 (ab53715, Abcam), Shh (ab53281, Abcam), Gli1 (ab49314, Abcam), Gli2 (ab167389, Abcam), and GAPDH (ab9484, Abcam) were used, individually.
5
Immunohistochemical Analysis of Glioma Markers
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Paraffin-embedded brain tissues were sectioned at 4-mm thickness. Xylene and ethanol of sequential concentrations was used for dewax and hydration. Antigen retrieval was performed using microwave for 20 min in 0.01 M citrate buffer (pH 6.0), followed by cooling to room temperature. After blocking by 3% H202 and then 10% FBS, samples were incubated with anti-Gli1 antibody (1:100; #ab49314; Abcam), anti-Ki67 antibody (1:500; #ab15580; Abcam), anti-Nestin antibody (1:250, #sc-43927, Santa Cruz), and anti-Sox2 antibody (1:400; #3579; CST) overnight at 4 °C and secondary antibody 30 min at room temperature. TUNEL staining was conducted using Colorimetric TUNEL Apoptosis Assay Kit (#C1098; Beyotime) based on the manufacturer’s instructions. Immunodetection was performed using DAB solution. Tissues were counterstained with hematoxylin.
6
Immunofluorescence Staining of GC Cells
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GC cells were incubated in chamber slides for 24 h, fixed in 4% paraformaldehyde for 20 min, and permeabilized with 0.1% Triton X-100 for 1 h. Next, cells were incubated with RAB31 (1:100, H00011031-M03; Abnova, Taipei, Taiwan) or GLI1 (1:100, ab49314; Abcam) primary antibody overnight at 4°C, after which the slides were washed three times. Finally, cells were incubated with goat anti-rabbit or anti-mouse IgG fluorescent secondary antibody (Thermo Fisher Scientific), and the nuclei were stained with DAPI. The cells were examined by confocal fluorescence microscopy at wavelengths of 488 and 594 nm.
7
Western Blot Analysis of Hedgehog Signaling
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Cell lysates were isolated in ice-cold RIPA buffer with complete EDTA-free Protease Inhibitor Cocktail (Roche, USA). The soluble fractions from lysates were collected by centrifugation at 17 000 g for 10 minutes at 4°C. The isolated protein was then separated on 4% to 20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the ethanol-pretreated polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with 5% nonfat dry milk in Tris-buffered saline with Tween 20 (TBST). Subsequently, the PVDF membrane was probed with rabbit anti-Ptch1 (1: 1000 dilution, AP06278PU-N, Origene, USA), rabbit anti-GlI1 (1: 1000 dilution, ab49314, Abcam, USA), rabbit anti-Hes-1 (1: 1000 dilution, ab71559, Abcam, USA) and anti-β-actin (1: 1000 dilution, sc-517582, Santa Cruz, USA) antibody in 5% bovine serum albumin in TBST at 4°C overnight. The PVDF membrane was then washed with TBST (3 times; 5 minutes each) and incubated with the secondary antibodies (goat anti-rabbit 1: 3000, ab6721, Abcam, USA) in 5% milk for 1 hour at room temperature. Membranes were then washed 3 times with TBST before being visualized using ECL Western Blotting Substrate Kit (Solarbio, Beijing, China). The β-actin was used as an internal control.
8
Western Blot Protocol for Liver Cancer
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The relative protein levels were analysed in this study by Western blotting using specific primary antibodies. Liver cancer cells were washed 3 times with PBS buffer and then subjected to total protein extraction. After boiling with protein loading buffer at 100°C for 5 minutes, approximately 25 g protein from each sample was separated by SDS‐PAGE, transferred onto PVDF membrane, blocked with 5% lipid‐free milk for 2 hours at room temperature, incubated with TBST‐containing primary antibodies for 1.5 hours, washed 3 times with TBST for 10 minutes, incubated with TBST containing secondary antibodies for 1 hours, washed 3 times again with TBST and finally developed with ECL solution (Amersham™). The primary antibodies used in this study against GLI1 (#ab49314), GLI3 (#ab126852), GAPDH (#ab8245), IL6 (#ab6672), phosphorylated JAK2 (#ab32101), phosphorylated STAT3 (#EP2147Y) and SOX2 (#ab97959) were purchased from the Abcam company. At least 3 independent biological repeats were performed for quantitation of protein abundances, and GAPDH was applied as the internal standard.
9
Antibodies for Molecular Signaling Analysis
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Mouse monoclonal antibody against mouse β-actin (ab8277), rabbit polyclonal antibody against mouse Gli1 (ab49314), rabbit monoclonal antibody against mouse β-catenin (ab32572), rabbit polyclonal antibody against mouse α-SMA (ab5694), rabbit monoclonal antibody against mouse Vimentin (ab92547), rabbit polyclonal antibody against mouse Collagen I (ab34710), rabbit polyclonal antibody against mouse Fzd9 (ab195718) and rabbit polyclonal antibody against mouse Fzd10 (ab137491) were purchased from Abcam (Cambridge, MA). Mouse monoclonal antibody against mouse Wnt10a was purchased from Santa Cruz (Beverly, MA). Rabbit monoclonal antibody against mouse Wnt7b was purchased from Bioss (Beijing, China).
10
Culturing Pancreatic Cancer Cell Lines
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The human PC cell lines Panc‐1 and BxPc‐3 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM supplemented with 10% foetal bovine serum (FBS) and 1% antibiotic/antimycotic (Life Technologies, Carlsbad, CA, USA). The cells were maintained at 37°C in a humidified 5% CO2 atmosphere. Antibodies against sHH (ab53281), SMO (ab5694), PTCH (ab53715), Gli‐1 (ab49314), GAPDH (ab8245) and EIF5A (ab32443) were purchased from Abcam (Cambridge, MA, USA). Recombinant sHH was obtained from R&D Systems (Minneapolis, MN, USA).
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