Cell lysates were subjected to polyacrylamide gel electrophoresis, and then blotted onto Immobilin-P polyvinylidene difluoride membranes (Millipore, Bedford, MA). Antibodies for cyclin D1 (catalog # 2978), CDK2 (catalog # 2546), phospho-Rb (catalog # 8516), Rb (catalog # 9309), PARP(catalog No # 9532), Caspase 3 (catalog # 9662), Bcl2 (catalog # 2876), BclXL (catalog # 2764), Bax (catalog No # 5023), CD44 (catalog # 3570), CD133 (catalog # 64326), phospho-STAT5 (catalog #s 4332, 9359), STAT5 (catalog # 25656), phospho-STAT3 (catalog # 9131), STAT3 (catalog # 4904), phospho-ERK (catalog # 4370) and ERK (catalog # 9102), ABCG2 (catalog #42078), DCLK1 (catalog # 62257) were purchased from Cell Signaling Technology (Danvers, MA, USA), for DCLK1 (catalog # ab37994), Oct-4 (catalog # ab189857) from Abcam Inc. (Cambridge, MA, USA), CDK4 (catalog # MA5–13498), CDK6 (catalog # MA5–13338) from Thermofisher (Waltham, MA, USA) and DCLK1 (catalog # SAB2420186) and Actin (catalog # A1978) from Sigma Aldrich and GAPDH (catalog # sc-365062) Santacruz Biotechnology Inc (Santa Cruz, CA, USA). Specific proteins were detected by chemiluminescence system.
Dclk1
Manufactured by Abcam
Sourced in United States
DCLK1 is a gene that encodes a type of serine/threonine-protein kinase. It is involved in the regulation of the cell cycle and cell division.
Automatically generated - may contain errors
Lab products found in correlation
Cyclin d1, by Cell Signaling Technology (3 mentions)
Epcam, by Abcam (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Alexa fluor 568 donkey anti goat igg, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488 donkey anti rabbit igg, by Thermo Fisher Scientific (3 mentions)
Notch1, by Cell Signaling Technology (2 mentions)
Cox 2, by Abcam (2 mentions)
β catenin, by Santa Cruz Biotechnology (2 mentions)
Phospho akt s473, by Cell Signaling Technology (2 mentions)
Fkbp12, by Abcam (2 mentions)
Phospho erk1 2t202 y204, by Cell Signaling Technology (2 mentions)
Phosstop phosphatase inhibitor, by Roche (2 mentions)
α tubulin, by Cell Signaling Technology (2 mentions)
Krasg12v, by Cell Signaling Technology (2 mentions)
Odyssey clx imager, by LI COR (2 mentions)
Erk1 2, by Cell Signaling Technology (2 mentions)
Complete protease inhibitor, by Roche (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
β catenin, by Cell Signaling Technology (2 mentions)
Abcg2, by Cell Signaling Technology (1 mentions)
15 protocols using dclk1
1
Protein Expression Analysis of Cellular Samples
2
Fixation and Immunohistochemical Analysis of Lung and Eye Tissues
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Freshly isolated lungs were perfused with Methacarnoy’s solution (60% absolute methanol, 30% chloroform, and 10% acetic acid), fixed in solution for 24 h, and then washed in 100% methanol before embedding in paraffin. Freshly isolated eyeballs were enucleated and fixed in Methacarnoy’s solution. 5-µm sections were stained with periodic acid–Schiff’s (PAS) reagent or subjected to immunohistochemical analysis with the following antibodies: Muc5b, generated by immunizing rabbits with synthetic peptides corresponding to the unique peptide sequence ELGQKVKCDVSSGLV, commercially available Muc5ac antibody (45M1; Sigma-Aldrich), or DCLK1 (Abcam; Ab31704). Bound primary antibody was detecting using the following antibodies; goat anti-rabbit IgG Alexa Fluor 488 (Invitrogen) and goat anti-mouse IgG Alexa Fluor 488 (Invitrogen). Sections were counterstained with DAPI. Images were captured using a Zeiss Axioimager.D2 upright microscope/Coolsnap HQ2 camera (Photometrics) through MetaVue Software. Specific bandpass filter sets for DAPI and FITC were used to prevent bleed-through from one channel to the next. Images were then processed and analyzed using ImageJ.
3
Multimarker Immunofluorescence Analysis
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The following antibodies were utilized: EpCAM, Ki-67, Dclk1 (all from Abcam, Cambridge, MA), Alexa Fluor 488 Donkey Anti-Rabbit IgG and Alexa Fluor® 568 Donkey Anti-Goat IgG (from Life Technologies, IL, USA).
4
Immunohistochemical Analysis of Lung and Colon Tissue
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Lungs were inflated and colons were flushed with PBS, then both tissues were fixed with 10% formalin, embedded in paraffin, cut into 5-μm sections, and adhered to charged slides. Slides were stained with H&E or left unstained. For IHC, lung sections were deparaffinized in CitriSolv (Fisher, 1601) and rehydrated in graded ethanol. Heat-mediated antigen retrival was performed with citrate-based Antigen Unmasking Solution (Vector labs, H3300). Sections were blocked and incubated with primary antibodies overnight at 4°C, and detected by Alexa Fluor 488- (Jackson ImmunoResearch Laboratories, 111–545-003) and 555- (Invitrogen, A21435) conjugated secondary antibodies. Slides were mounted using DAPI-containing mounting media (Vector labs, H-1000). Images were obtained with immunofluorescence microscopy (BX-51; Olympus) and analyzed with SPOT Advanced software (Diagnostic Instruments). Antibodies used: SQSTM1/p62 (Progen, GP-P62 C), CTNNB1 (Sigma, HPA029160), MKI67/Ki67 (eBioscience, SolA15), DCLK1 (Abcam, ab31704).
5
Molecular Profiling of Intestinal Stem Cells
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We used the following antibodies: Dclk1, Lgr5, Bmi1, Hes1, Tcf4, Cox1, Cox2, EpCam, CD45, CD31 (all from Abcam, Cambridge, MA), CXCL1, CyclinD1, cMYC, β − catenin (Santa Cruz Biotechnology, USA), Notch1, NfkB-p65, CyclinD1, Ras, β-actin (Cell Signaling, Danvers, MA, USA), anti-rabbit IgG, anti-mouse IgG, anti-goat IgG (Jackson ImmunoResearch, West Grove, PA, USA), Alexa Fluor® 488 donkey anti-rabbit IgG, and Alexa Fluor® 568 donkey anti-goat IgG (Invitrogen, USA).
6
Immunohistochemical and Immunofluorescence Analyses of FFPE Tumor Samples
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For immunohistochemistry analyses, samples were fixed overnight in 4% formalin solution and embedded in paraffin. In brief, 3-µm-thick sections of FFPE tumors were deparaffinized and antigen retrieval was performed by boiling the sections in citrate buffer at pH 6.0 or EDTA at pH 9.0 for 20 min. Primary antibodies used were as follows: Dclk1 (1:50; pH 6.0; Abcam), Sox9 (1:50; pH 6.0; Millipore), Ki67 (1:50; pH 6.0; Cell Marque), corresponding secondary antibody detection kits for reduced background on murine tissue were used (Histofine Simple Stain Mouse MAX PO; Medac) and stained on an automated stainer (LabVision Autostainer 480S; Thermo Fisher Scientific). All slides were scanned with a Panoramic 250 slide scanner (3D Histech.com).
For immunofluorescence, frozen sections were incubated in the target retrieval solution (Dako) for 7 min. Primary antibodies are listed in the Cell lines and reagents section. Slides were analyzed using a sp5 confocal microscope (Leica).
For immunofluorescence, frozen sections were incubated in the target retrieval solution (Dako) for 7 min. Primary antibodies are listed in the Cell lines and reagents section. Slides were analyzed using a sp5 confocal microscope (Leica).
7
Immunoblotting of Protein Lysates
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Cell lines were lysed in RIPA buffer supplemented with cOmplete protease inhibitors (Roche), PhosSTOP phosphatase inhibitors (Roche), and 0.1% benzonase (Novagen) on ice for 60 minutes. Lysates were clarified by centrifugation at 20,000 x g for 10 minutes at 4 °C and immunoblotting was performed using an Odyssey CLx Imager (LI-COR) as previously described.26 (link) The following primary antibodies were employed in this study: HA (Cell Signaling, #3724 and #2367), DCLK1 (Abcam, #ab31704), phospho-ERK1/2 T202/Y204 (Cell Signaling, #4370), ERK1/2 (Cell Signaling, #4696), phospho-AKT S473 (Cell Signaling, #4060), AKT (Cell Signaling, #2920), FKBP12 (Abcam, #ab24373), KRASG12V (Cell Signaling, #14412) and α-Tubulin (Cell Signaling, #3873). Fluorescently labelled infrared secondary antibodies (Licor, IRDye) were employed as appropriate.
8
Analyzing Stem Cell Markers in Colon Cancer
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Human colon
cancer HT-29 cells,
grown in spheroid or monolayer condition were treated with vehicle
or NSGMs for 24 h, were trypsinized and single cells were resuspended
at 106 cells/mL in PBS buffer. Cells were incubated with
fluorophore conjugated antibody for 30 min at 4 °C and washed
once with PBS buffer prior to analysis. Following antibody and dilution
were used: LGR5-PE) (Dilution 1:50, Origene), DCLK1(Dilution 1:33,
Abcam). Cell sorting was performed using FACSAria II High-Speed Cell
Sorter (BD Biosciences) and data were analyzed with FCS Express 4
Flow Cytometry software (De-Novo Software).
cancer HT-29 cells,
grown in spheroid or monolayer condition were treated with vehicle
or NSGMs for 24 h, were trypsinized and single cells were resuspended
at 106 cells/mL in PBS buffer. Cells were incubated with
fluorophore conjugated antibody for 30 min at 4 °C and washed
once with PBS buffer prior to analysis. Following antibody and dilution
were used: LGR5-PE) (Dilution 1:50, Origene), DCLK1(Dilution 1:33,
Abcam). Cell sorting was performed using FACSAria II High-Speed Cell
Sorter (BD Biosciences) and data were analyzed with FCS Express 4
Flow Cytometry software (De-Novo Software).
9
Immunohistochemical Profiling of Premalignant Tumors
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At least 15 premalignant tumors per group were used to perform immunostaining experiments as described earlier [5 (link)] in Figure 1 , Figure 2 , Figure 3 and Figure 4 . The slides were prepared for immunostaining using deparaffinization, rehydration, and antigen retrieval. Finally, immunostainings were performed using a SuperPictureTM 3rd Gen IHC detection kit (Cat#87-9673; Invitrogen, Camarillo, CA) as per the manufacturer’s instruction. Briefly, the samples were blocked for endogenous peroxidase activity followed by incubation in protein-blocking buffer for 20 min. Tissue samples were then washed and incubated overnight at 4 °C with primary antibodies including ALDH1 (Cat#ab23375, Abcam, Cambridge, MA, dilution 1:100), BMI1 (Cat#NBP18732, Novus Biologicals, Littleton, CO, dilution 1:100), CD133 (Cat#PAB12663, Abnova, Taipei, Taiwan, dilution 1:100), DCLK1 (Cat#ab37994, Abcam, dilution 1:25), LGR5 (Cat#CF503316, Origene, Rockville, MD, dilution 1:100), MSI1 (cat#ab52865, Abcam, dilution 1:100), and β-catenin (Cat#9587S, Cell Signaling Technology, Danvers, MA, dilution 1:200). After multiple washes, samples were incubated with HRP-conjugated secondary antibody for 30 min, and signals were detected using 3,3’Diaminobenzidine (DAB) chromogen. Finally, sections were counterstained with hematoxylin, followed by permanent mounting and microscopic imaging.
10
Antibody Panel for Intestinal Stem Cell Markers
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The following antibodies were used: Dclk1, BrdU, Lgr5, Bmi1, Msi1, COX1, COX2, Claudin1, Claudin5, Claudin7 [Abcam, Cambridge, MA]; Claudin-7, E-Cadherin, β-actin, Notch1, NFkB, Betacatenin, [Cell Signaling, Danvers, MA]; Alexa Fluor® 488 donkey anti-rabbit IgG (A-21206), Alexa Fluor® 568 Donkey Anti-Goat IgG (A-11057), Hoechst 33342 DNA stain (H1399) [Invitrogen].
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