Total RNA was isolated from exponentially growing cells (70% confluence) using RNeasy mini Kit (Qiagen, Netherlands) according to by manufacturer's instruction. Real-time quantitative PCR (qPCR) was also performed to analyse mRNA expression levels [16 (link)]. Primer used for RT-PCR and qPCR are as previously described [81 (link)]. Protein expression was analysed using western blot by using the following antibodies: TFF3 (ab108599, 1:1000, Abcam); phospho-AKT1 S473 (ab66138,1:2000, Abcam); pan-AKT (ab8805, 1:2000, Abcam); β-Actin (sc-47778, 1:10000, Santa Cruz), BCL-2 (sc-509, 1:5000, Santa Cruz), Bax (sc-70407, 1:5000, Santa Cruz). The secondary anti-rabbit and anti-mouse horseradish peroxidase (HRP)-conjugated antibodies were purchased from Cell Signaling Technology. Proteins were visualized using the chemiluminescence ECL kit (SuperSignal West Pico substrate; Pierce, Rockford, IL) and read on ImageQuant system LAS500 (GE Healthcare).
Ab108599
Manufactured by Abcam
Sourced in United States
Ab108599 is a monoclonal antibody that recognizes the human GAPDH protein. GAPDH is a glycolytic enzyme involved in energy production within cells. This antibody can be used for the detection of GAPDH in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab11197, by Abcam (2 mentions)
Mab1501, by Merck Group (2 mentions)
Na931v, by GE Healthcare (2 mentions)
A11017, by Thermo Fisher Scientific (2 mentions)
Mu392a uc, by BioGenex (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Ab8805, by Abcam (1 mentions)
Ab66138, by Abcam (1 mentions)
Bcl 2 sc 509, by Santa Cruz Biotechnology (1 mentions)
β actin sc 47778, by Santa Cruz Biotechnology (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (1 mentions)
Ab215711, by Abcam (1 mentions)
M2 616, by BD (1 mentions)
Sc 393925, by Santa Cruz Biotechnology (1 mentions)
A11070, by GE Healthcare (1 mentions)
A11072, by GE Healthcare (1 mentions)
Na934v, by Santa Cruz Biotechnology (1 mentions)
Sc 2350, by Santa Cruz Biotechnology (1 mentions)
6 protocols using ab108599
1
RNA Extraction and qPCR Analysis
2
Immunoblotting and Immunofluorescence Assays
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In the present study, mouse monoclonal anti-MUC2 (ab11197, 1/500 WB and 1/100 IF, Abcam), rabbit monoclonal anti-TFF3 (ab108599, 1/1500 WB, Abcam), mouse monoclonal anti-SI ([53]; HSI-4/34 or Caco-3/73, 1/100 WB), rabbit monoclonal anti-DPPIV or CD26 [EPR20819] (ab215711, 1/2000 WB, Abcam), rabbit monoclonal anti-YAP/TAZ (D24E4, 1/1500 WB, Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti-YAP (D8H1X, 1/1000 WB, 1/150 IF, Cell Signaling Technology), mouse monoclonal anti-TAZ (M2-616, 1/300 IF, BD Biosciences, NJ, USA) mouse monoclonal anti-CDX2-CD88 (MU392A-UC, 1/700, BioGenex, Freemont, CA, USA), mouse monoclonal anti-HNF1α [F-7] (sc-393925, 1/300 WB, Santa Cruz Biotechnology, Dallas, TX, USA), goat polyclonal anti-HNF4α [C-19] (sc-6556, 1/600 WB, Santa Cruz Biotechnology) and anti-β-actin (MAB1501, 1/20,000, Millipore, Etobicoke, ON, Canada) were used as primary antibodies. In addition, AlexaFluor 488 or 594 goat anti-mouse (A11017, A11072, 1/400; Thermo Fisher Scientific, Ottawa, ON, Canada) and goat anti-rabbit (A11070, A11072, 1/400), ECL HRP-linked anti-mouse (NA931V, 1/4000, GE Healthcare, Mississauga, ON, Canada) and anti-rabbit (NA934V, 1/4000) and HRP-linked bovine anti-goat (sc-2350, 1/4000, Santa Cruz Biotechnology) were used as secondary antibodies.
3
Characterization of Intestinal Epithelial Markers
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The primary antibodies used in this study include mouse monoclonal anti-MUC2 (Abcam, ab11197, 1/500 WB and 1/100 IF), rabbit monoclonal anti-TFF3 (ab108599, 1/1500 WB, Abcam, Toronto, ON, Canada), mouse monoclonal anti-SI ([53 (link)]; HSI-4/34 or Caco-3/73, 1/100 WB), rabbit polyclonal anti-DPPIV or CD26 (ab129060, 1/2000 WB, Abcam), mouse monoclonal anti-DPPIV (ab3154, 1/100 WB, Abcam), mouse monoclonal anti-DPPIV ([54 (link)] DAO 7/219, 1/100 IF, a gift from A. Quaroni), rabbit monoclonal anti-YAP/TAZ (D24E4, 1/1500 WB, 1/50 IF, Cell Signaling, Danvers, MA, USA), mouse monoclonal anti-CDX2-CD88 (MU392A-UC, 1/700, BioGenex, Freemont, CA, USA), mouse monoclonal anti-LGR5 (UMAB212, 1/100 WB and 1/100 IF, Origene, Rockville, MD, USA) and anti-β-actin (MAB1501, 1/20,000, Millipore, Etobicoke, ON, Canada). The secondary antibodies used in this study include AlexaFluor 488 or 594 goat anti-mouse (A11017, A11072, 1/400, and goat anti-rabbit (A11070, A11072, 1/400; Thermo Fisher Scientific, Ottawa, ON, Canada), ECL HRP-linked anti-mouse (NA931 V, 1/4000, GE Healthcare, Mississauga, ON, Canada) and anti-rabbit (NA934 V, 1/4000).
4
Protein Extraction and Immunoblotting Protocol
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Total cellular and tissue protein was extracted using RIPA lysis buffer containing protease and phosphatase inhibitors (Thermo Fisher, USA). Samples were incubated for 20 min on ice after sonication with an ultrasonic cell crusher and then centrifuged for 20 min at 13,000 rpm at 4°C. Protein concentrations were determined using the Pierce BCA protein assay kit (Thermo Fisher, USA). Immunoprecipitation was performed using Pierce Protein A/G Magnetic IP/Co-IP Kit (Thermo Fisher, USA) following standard protocol. Lysate samples (40 μg) were separated with 12% SDS-PAGE and transferred onto PVDF membranes (Millipore, USA). Membranes were incubated with primary antibodies overnight at 4°C. Membranes were washed with TBST and incubated for 2 h with HRP-conjugated anti-rabbit secondary antibodies (1:5000; CST, USA), followed by detection and visualization using ECL Western blotting detection reagents (Pierce, Thermo Fisher, USA). The primary antibodies used were anti-TFF3 (1:1000; ab108599, Abcam, USA), anti-HIF-1α (1:200; ab1; Abcam, USA), anti-MMP2 (1:1000; ab86607; Abcam, USA), anti-AKT1 (phospho S473) (1:1000; ab81283; Abcam, USA), anti-cleaved caspase-3 (1:1000; #9664; CST, USA), anti-cleaved caspase-9 (1:1000; #7237; CST, USA)
5
Immunohistochemical Analysis of FFPE Tissues
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3–5 µm tissue sections of formalin‐fixed and paraffin‐embedded (FFPE) tissue were used. Immunostainings were performed on the BenchMark XT immunostainer (Ventana Medical Systems), using CC1 mild buffer or Ultra CC1 buffer (Ventana Medical Systems) for 30 min at 100°C, and using antibodies rabbit anti‐TFF3 (1:250, Abcam, ab108599), mouse anti‐FABP1 (1:1,000, Abcam, ab7366), rabbit anti‐OLFM4 (1:100, Atlas Antibodies, HPA077718), mouse anti‐EPCAM (1:100, Thermo Scientific, MS‐144‐P1), rabbit anti‐Ki67 (1:400, Abcam, ab16667), mouse anti‐Ki67 (1:50, Dako, M7240), rabbit anti‐LYZ (1:1500, Abcam, ab108508), rabbit anti‐EREG (1:50, Thermo Fischer Scientific, PA5‐24727), anti‐PARP1, mouse anti‐MUC2 (1:50, Leica, NCL‐MUC‐2), mouse anti‐CK17 (1:10, Dako, M7046), and mouse anti‐MMP7 (1:100, Thermo Fisher Scientific, MA5‐14215). Images were taken using AxioVert.A1 (Zeiss) or CQ1 (Yokogawa) microscopes or scanned using the Pannoramic SCAN 150 scanner (3DHISTECH).
6
Quantitative Western Blot Analysis
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Western blot analysis was performed as early reported18 Proteins were incubated with antibodies against TFF3 (1:1000; ab108599; Abcam, Cambridge, MA, USA), C/EBPβ (sc‐150; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Vimentin (1:1000, ab92547), E‐cadherin (1:50, ab1416) and GAPDH (1:2500; ab9485, Abcam), followed by incubation with the secondary antibody, horseradish peroxidase‐labelled goat anti‐rabbit IgG (1:2,000; A0208; Beyotime Institute of Biotechnology, Shanghai, China). Each sample was repeated in triplicate. ImageJ v1.48u software (National Institutes of Health, Bethesda, Maryland, USA) was utilized for quantification analyses.
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