The total protein of tissues and cells was extracted. After ice bathing and centrifugation, the sample was added to each lane for electrophoresis separation. The separated protein was transferred onto a nitrocellulose membrane. Then, the nitrocellulose membrane was blocked with 5% skim milk powder at 4°C overnight and incubated overnight with primary rabbit anti‐mouse polyclonal antibodies diluted at the ratio of 1:1000: NEK2 (ab115731), β‐catenin (ab16051), Nanog (ab21624), Oct‐4 (ab19857), CXCL16 (ab101404), EGFR (ab52894), TCF‐4 (ab217668) and Pygo2 (ab109001) and rabbit anti‐mouse monoclonal antibodies diluted at 1:1000: CD24 (ab64064), and CD44 (ab51037), all of which were purchased from Abcam Inc, Cambridge, UK. The membrane was washed 3 times with PBS at room temperature, 5 minutes for each, incubated with oscillation with the addition of the secondary antibody of horseradish peroxidase (HRP)‐labelled goat anti‐rabbit immunoglobulin (IgG) (1:10 000, ab6728, Boster Biological Technology Co. Ltd, Wuhan, China) at 37°C for 1 hour, washed with Tris‐buffered saline‐Tween (TBST) and visualized using HRP electroluminescence (ECL). Grey value analysis of the target bands was performed using ImageJ software, and the experiment was repeated three times independently.
Ab217668
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab217668 is a primary antibody targeting the human ABCC1 protein. It is produced in rabbit and purified by protein A affinity chromatography.
Automatically generated - may contain errors
Lab products found in correlation
Ab32072, by Abcam (3 mentions)
Ab32572, by Abcam (3 mentions)
Ab137872, by Abcam (2 mentions)
Ab19857, by Abcam (1 mentions)
Ab115731, by Abcam (1 mentions)
Ab109001, by Abcam (1 mentions)
Ab16051, by Abcam (1 mentions)
Ab101404, by Abcam (1 mentions)
Ab52894, by Abcam (1 mentions)
Ab21624, by Abcam (1 mentions)
Cd24 ab64064, by Abcam (1 mentions)
Specific secondary antibody, by Abcam (1 mentions)
Ab106629, by Abcam (1 mentions)
Ab6302, by Abcam (1 mentions)
Anti β actin, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Immobilon forte western hrp substrate, by Merck Group (1 mentions)
Ab6046, by Abcam (1 mentions)
12 protocols using ab217668
1
Protein Expression Analysis of Stem Cells
2
Western Blot Analysis of LMX1A, β-catenin, and TCF4
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The collected cells were lysed in PMSF buffer. The protein concentration was determined by using a BCA protein assay kit (Beyotime, China). Protein was separated by SDS-PAGE electrophoresis and transferred to membranes; membranes were subsequently blocked with 5% bovine serum albumin (BSA) at room temperature (RT) for 2 h. Next, the membranes were incubated with anti-LMX1A (ab106629; Abcam, Cambridge, MA, USA), anti-β-catenin (ab6302; Abcam), anti-TCF4 (ab217668; Abcam), and anti-β-actin (Abcam) overnight at 4℃. After washing, the membrane was then probed with specific secondary antibodies (Abcam) at room temperature for 2 h, and ECL visualization was performed. The protein bands were visualized and analyzed using Versa Doc™ imaging system.
3
Immunoblot Analysis of Protein Samples
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Protein samples were harvested from cells using the SDS sample buffer (100 mM Tris-HCl, pH 6.8, 2% SDS, 40% glycerol, 5% β-mercaptoethanol, and 0.1% bromophenol blue). Samples were heated at 99°C for 10 min prior to being subjected to the immunoblot analysis. The protein samples were then transferred to a PVDF membrane (IPVH00010, pore size 0.45 μm, Merck Millipore). The membrane was blocked using 5% nonfat milk at 25°C for 1 h, followed by the incubation of primary antibodies at 4°C for 16 h. Primary antibodies used were anti-TCF4 (ab217668, 1:1,000, abcam), anti-INTU (ab229243, 1:1,000, abcam), anti-IFT88 (13967-1-AP, 1:1,000, Proteintech) and anti-β-TUBULIN (ab6046, 1:2,000, abcam). The membrane was washed three times with 1× TBST each for 10 min, before being subjected to the incubation of secondary antibodies at 25°C for 1 h. Secondary antibodies used were HRP-conjugated goat anti-rabbit IgG (H + L) (11-035-045, 1:5,000) and HRP-conjugated goat anti-mouse IgG (H + L) (115-035-062, 1:10,000) from Jackson ImmunoResearch. The membrane was washed three times with 1× TBST each for 10 min, prior to the detection of chemiluminescent signal. The signal was developed using Immobilon Forte Western HRP substrate (WBLUF0100, Merck Millipore), and the images were captured and processed using ChemiDoc™ Touch Imaging System (170-8370, Bio-Rad).
4
Chromatin Immunoprecipitation (ChIP) Assay
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ChIP was performed using a Pierce Magnetic ChIP kit (Cat. NO.: 26157, Thermo Fisher, USA) according to the manufacturers’ instructions. In brief, 4 × 106 cells were used in the experiment. Crosslinked chromatin was broken into 150- to 1000-bp fragments by MNase digestion. The chromatin was then immunoprecipitated with 2 μg of rabbit anti-TCF4 antibody (Abcam ab217668) or 2 μg of rabbit IgG with rotation overnight at 4 °C. Then, 20 μl of magnetic beads were added into each tube, and the tubes were incubated at 4 °C for 2 h with mixing. The magnetic beads were washed with IP wash buffer five times and then eluted. After purification, the immunoprecipitated chromatin was analyzed by semiquantitative-PCR and qRT-PCR. For semiquantitative-PCR, the expression levels of the DNA products were detected through 1.5% agarose gel electrophoresis. Primer pairs used in PCRs were designed within the promoter region (Table S7 , Fig. 5A ).
5
Transcriptional Regulation and Self-Renewal Assay
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Total mRNA was reverse‐transcribed into cDNA (AT301 TransGen Biotech, Beijing, China), and real‐time quantitative PCR was performed with CFX96 Real‐Time PCR Detection System (Bio‐Rid, Hercules, CA, USA). For western blot, the protein from cell extracts was separated by 10% SDS‐PAGE electrophoresis and was later transferred onto PVDF membrane. Membranes were incubated with ESR1 (1:3000, EPR4097, ab108398; Santa Cruz, Dallas, TX, USA), TCF4 (1:2000, ab217668; Abcam, Cambridge, MA, USA), CMYC (1:2000, ab32072; Abcam), LEF1 (1:2000, ab137872; Abcam), WNT1 (1:1000, ab15251; Abcam), CTNNB1 (1:1000, C2206; Sigma‐Aldrich, St. Louis, MO, USA), VINCULIN (1:5000, #18799; Cell Signaling Technology, Danvers, MA, USA), and then detected using ECL Blotting Detection Reagents (Merck Millipore, Burlington, MA, USA). Cells of different groups were suspended in DMEM/F12 Medium supplemented with 20 ng/mL EGF (BD Biosciences, San Jose, CA, USA), bFGF and 4 μg/mL insulin (Sigma), and then plated in 6‐well ultra‐low attachment dishes (1000 cells/mL; Corning Incorporated, Tewksbury, MA, USA). To analyse the self‐renewal ability, sphere number of each captured image was counted by using phase contrast microscope (Nikon, Tokyo, Japan).
6
ChIP Analysis of INTU and IFT88 Promoters
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The chromatin immunoprecipitation (ChIP) assay was performed using Pierce™ Magnetic ChIP Kit (26157, Thermo Fisher Scientific). The experimental procedures were carried out following the manufacturer’s instructions. Two micrograms of anti-TCF4 antibody (ab217668, abcam) were used for the immunoprecipitation, while the same amount of normal rabbit IgG (I-1000, Vector Laboratories, lnc.) was used as negative control. Twenty nanograms of recovered genomic DNAs from each of input, normal rabbit IgG and TCF4 immunoprecipitated samples were used in the following real-time PCR to analyze the levels of INTU and IFT88 promoter fragments. The primers used were INTU promoter-F, 5′-CAGCCTGGACTTCGCGAG-3′; INTU promoter-R, 5′-TGAAGGCGGTGGTGTCAG-3′; IFT88 promoter-F, 5′-AAAACGGACACCTTAAGCGC-3′ and IFT88 promoter-R, 5′-CTTGTGAACCTTGGAAGCCC-3′.
7
Wnt/β-catenin Signaling Pathway Analysis
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Cells were cultured in 6-well plates at 2×106/well and transfected with Wnt10a-siRNA fragments or treated with 0.4 nM LGK-974, a specific inhibitor for porcupine homolog (Drosophila; PORCN), in Wnt/β-catenin signaling pathway. After 48 h of incubation at 37°C, total protein was extracted using iced lysis buffer (1% Triton X-100, 50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 0.1% SDS; 1 mM phenylmethanesulfonyl fluoride; 1 mM EDTA). Subsequently, the total protein concentration was determined using a bicinchoninic acid assay (BCA Protein Assay kit; Generay Biotech Co., Ltd., Shanghai, China). A total of 10 µg protein was separated on an 10% SDS-PAGE gel, transferred to nitrocellulose membranes followed by blocking with 5% non-fat milk for 2 h at room temperature, and then incubated overnight with primary mouse monoclonal antibodies against β-catenin (1:250, sc65480; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), cyclin D1 (1:400, sc-70899; Santa Cruz Biotechnology, Inc.), LEF1 (1:800, ab137872), TCF4 (1:500, ab217668) and GAPDH (1:2,000, ab181602) (Abcam, Cambridge, MA, USA) at 4°C. Membranes were subsequently washed and incubated with a horseradish peroxidase-conjugated secondary antibody (1:10,000, ab97051) for 1 h at 25°C. Immunoreactivity was determined using an enhanced chemiluminescence kit (Merck KGaA).
8
Immunohistochemical Analysis of Brain Slices
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Animals were anaesthetised with halothane followed by transcardial perfusion with PBS and 4% PFA. The brains were fixed in 4% PFA overnight followed by sectioning on Leica Vibratome to obtain 30 µm sections. Some animals were taken for dual experiments, where half brain was used for immunostaining. In these cases, half brains were directly fixed in 4% PFA for two overnights at 4 °C without transcardial perfusion. The floating sections were first blocked in 10% normal goat serum, 1% BSA, 0.1% triton and 100 mM glycine for 1 h at RT and stained with primary antibody (in 1% normal goat serum, 0.1% BSA, 0.1% triton and 100 mM glycine) overnight at 4 °C. Sections were then washed and incubated with fluorophore conjugated secondary antibody for 1 h at RT in dark, before mounting and imaging. Primary antibodies used were: CaMKII alpha (Invitrogen #13-7300), calbindin (SySy #214 005), parvalbumin (SySy #195 004), cFos (SySy #226 004), NeuN (Millipore # MAB377) and TCF4 (Abcam #ab217668).
9
Western Blot Analysis of Wnt Pathway
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All proteins were extracted from cells using RIPA lysis buffer (Beyotime Institute of Biotechnology). Then, the concentrations of proteins were measured by Bicinchoninic Acid Protein Assay Kit (Pierce Biotechnology, Inc.) following the protocol. Afterward, 20 μg proteins were separated by 10% SDS‐PAGE gel and transferred onto PVDF membranes. Membranes were blocked with 5% skimmed milk at room temperature for 50 min and incubated with primary antibodies at 4°C overnight. The primary antibodies were as follows: rabbit anti‐β‐catenin (ab32572; 1:5000; Abcam), rabbit anti‐TCF4 (ab217668; 1:10,000; Abcam), rabbit anti‐Cyclin D1 (ab16663; 1:100; Abcam), rabbit anti c‐Myc (ab32072; 1:1000; Abcam), and rabbit anti GAPDH (ab9485; 1:2500; Abcam). After that, membranes were washed with Tris‐buffered saline solution (containing 0.1% Tween‐20) and then incubated with goat anti‐rabbit IgG H&L secondary antibody (ab7090; 1:1000; Abcam) at room temperature for 50 min at dark. Next, proteins were detected with enhanced chemiluminescence (Pierce Biotechnology, Inc.) and analyzed using ImageJ 1.48 software (National Institutes of Health) while normalized to GAPDH.
10
Immunohistochemical Profiling of Tumor Angiogenesis
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All paraffin embedded specimens were immobilized in 10% formalin. All sectioned specimens (4 μm thick) were waxed, dehydrated with a gradient alcohol series, and washed in PBS (pH 7.2) for 10 min. Sections were incubated with 3% H2O2 methanol at room temperature for 10 min to inhibit endogenous peroxidase activity. Samples were boiled in 1.0% citrate (pH 6.0) for 2 min at high pressure for antigen retrieval. Slides were washed in PBS and blocked in goat serum at room temperature for 20 min. Sections were probed with rabbit monoclonal anti-Foxm1(1:250, ab207298, Abcam, USA), anti-β-catenin (1:100, ab32572, Abcam, USA), anti-Tcf4 (1:150, ab217668, Abcam, USA), anti-E -cadherin (1:200, ab1416, Abcam, USA), and anti-CD34 (1:200, ab762, Abcam, USA) primary antibodies overnight at 4 °C, followed by the appropriate secondary antibodies for 30 min. All CD34 labeled specimens were stained with PAS to characterize the glycolic basement membrane of the vascular endothelial cells, in addition to vascular-like (VM) channels [24 (link), 25 (link)]. After rinsing with PBS, freshly prepared 3, 3 ‘-diaminobenzidine (DAB) solution was added to the sections for 5 min, and hematoxylin was back-dyed, dehydrated, air-dried and loaded.
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