For Western blotting, anti-β-actin, anti-CyclinD1, anti-CDK2 (Cell Signaling, Danvers, MA, USA), anti-SMAD4, anti-p21/Cip1 (Abcam, Cambridge, MA, USA), anti-KLF4 (Proteintech, Wuhan, Hubei, China), anti-proliferating cell nuclear antigen (anti-PCNA) (Proteintech, Wuhan, Hubei, China), and anti-myeloid-associated differentiation marker (anti-Myadm) (Novus, Littleton, CO, USA) antibodies were used as the primary antibodies. The signals were detected using the enhanced chemiluminescence detection method and were quantified by densitometry.
Anti klf4
Manufactured by Proteintech
Sourced in United States
Anti-KLF4 is a laboratory reagent used for the detection and quantification of the Krüppel-like factor 4 (KLF4) protein in biological samples. KLF4 is a transcription factor involved in various cellular processes, including stem cell maintenance, cell differentiation, and proliferation. This antibody can be used in techniques such as Western blotting, immunohistochemistry, and immunocytochemistry to study the expression and localization of the KLF4 protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti myadm, by Novus Biologicals (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Anti cdk2, by Cell Signaling Technology (1 mentions)
Anti cyclin d1, by Cell Signaling Technology (1 mentions)
Anti smad4, by Abcam (1 mentions)
Lsm 510 meta confocal laser scanning microscope, by Zeiss (1 mentions)
Anti srf, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Glass bottomed cell culture dishes, by NEST Biotechnology (1 mentions)
Anti sirt1, by Santa Cruz Biotechnology (1 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)
Anti p21, by Abcam (1 mentions)
Arrayscan vti hcs reader, by Thermo Fisher Scientific (1 mentions)
Anti jagged1, by Abcam (1 mentions)
Anti oct4, by Proteintech (1 mentions)
Anti dll1, by Abcam (1 mentions)
Anti notch1 icd, by Abcam (1 mentions)
Anti jagged2, by Abcam (1 mentions)
Anti dll4, by Abcam (1 mentions)
9 protocols using anti klf4
1
Western Blot Protein Expression Analysis
2
Immunofluorescence Staining of Transfected HASMCs
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At 24 h post-transfection and relative stimulation, walled HASMCs were digested using trypsin, centrifuged and resuspended, and then plated on glass-bottomed cell culture dishes (NEST, Cat#801001). After serum deprivation overnight, cells were rinsed quickly in ice-cold PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. The permeabilization was achieved by 1% Triton X-100 in PBS for 10 min and rinsed with PBS, and cells were then blocked using 1% BSA in PBS for 30 min. The primary antibodies were incubated with cells at 4 °C overnight (anti-KLF4: Proteintech, Cat#11880-1-AP, 1:50; anti-SRF: Santa Cruz, Cat#sc-25290, 1:50). Secondary antibodies (Alexa Fluor 594-conjugated Goat anti-Rabbit IgG: Invitrogen, Cat#A-11012, 1:1000; Alexa Fluor 488-conjugated Goat anti-Mouse IgG: Invitrogen, Cat#A-11029, 1:200) were then incubated for 1 h at room temperature. Cells were mounted in a mounting medium with DAPI (Abcam, Cat#ab104139), and images were acquired with a Zeiss LSM 510 META Laser Scanning Confocal Microscope.
3
Immunoprecipitation and Western Blot Analysis
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The protein lysates, which obtained as aforementioned, were incubated with 1 µg IgG (Beyotime Institute of Biotechnology) and 20 µl resuspended Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Inc.) for 30 min at 4°C, then centrifugation was used to remove the non-specific protein adhering to protein A/G at 4°C at 2,500 × g for 5 min. After collecting and measuring the concentration of protein by the bicinchoninic acid method, the supernatants were made up to a total amount of 500 µl with IP buffer. The corresponding primary antibody with 5 µl was added to the samples including anti-KLF4 (ProteinTech Group, Inc.; cat. no. 11880), or anti-SIRT1 (Santa Cruz Biotechnology, Inc.; cat. no. sc-15404) or IgG (Beyotime Institute of Biotechnology) which as a contrast, followed by incubation overnight at 4°C. Subsequently, the samples were incubated with 100 µl Protein A/G PLUS-Agarose at 4°C for 4 h. Finally, the result was produced by western blot analysis.
4
Immunofluorescence Analysis of p21 and KLF4
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Cell immunofluorescence analyses were performed using anti-p21(Abcam, Cambridge, MA, US), and anti-KLF4 (Proteintech, Wuhan, China) as primary antibodies, followed by FITC (green fluorescence, Chemicon, Temecula, CA, USA)-conjugated secondary antibodies and visualized by Cellomics array scan (ArrayScan VTI HCS Reader; Cellomics). The nuclei were visualized by DAPI staining.
For the tissue immunofluorescence staining, sections were incubated with anti-SM-α actin (SMA, 1:50, Santa Cruz, Texas, USA) and anti-Myadm (1:50, Novus, Littleton, CO, USA) antibodies, followed by fluorescein-conjugated secondary antibodies (1:300 dilution). The cell nuclei were stained with DAPI.
For the tissue immunofluorescence staining, sections were incubated with anti-SM-α actin (SMA, 1:50, Santa Cruz, Texas, USA) and anti-Myadm (1:50, Novus, Littleton, CO, USA) antibodies, followed by fluorescein-conjugated secondary antibodies (1:300 dilution). The cell nuclei were stained with DAPI.
5
Western Blot Analysis of Cellular Proteins
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Cells were lysed in cell lysate, and then centrifuged at 12,000 × g for 20 min at 4 °C. The supernatant was collected and denatured. Proteins were separated in 10% SDS-PAGE and blotted onto polyvinylidene difluoride membrane (PVDF). The PVDF membrane was treated with TBST containing 50 g/L skimmed milk at room temperature for 4 h, followed by incubation with the primary antibodies: anti-CTR2 (1:200, Novusbio), anti-BCL-2 (1:500, Immunoway), anti-OCT-4 (1:1000, Proteintech), anti-KLF4 (1:500, Proteintech), anti-MDR1 (1:200, Santa),, anti-Notch1 ICD (1:1000, Abcam), anti-Notch2 ICD (1:1000, Abcam), anti-Jagged1 (1:500, Abcam), anti-Jagged2 (1:1000, Abcam), anti-DLL1 (1:500, Abcam), anti-DLL4 (1:500, Abcam), anti-Notch ICD (1:1000, Cell Signaling), anti-SDF-1 (1:1000, Abcam), anti-CXCR4 (1:2000, Abcam) and anti-β-actin (1:1000, Cell signaling) respectively, at 37 °C for 1 h. Membranes were rinsed and incubated for 1 h with the correspondent peroxidase-conjugated secondary antibodies. Chemiluminent detection was performed with the ECL kit (Pierce Chemical, Rockford, IL, USA). The amount of the protein of interest, expressed as arbitrary densitometric units, was normalized to the densitometric units of ß-actin.
6
Antibody-Based Protein Detection Protocol
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The antibodies specifically against SOX2, c-MYC, 14-3-3, and β-Catenin were purchased from Abcam (UK); anti-CD44 and anti-KLF4 were products of Proteintech (USA); anti-β-actin, HRP-conjugated anti-mouse or anti-rabbit IgG were purchased from ZSGBBIO (CHN); anti-CBY1 was purchased from Santa Cruz (USA); anti-SP3 was purchased from Sigma-Aldrich (USA); anti-Nanog, anti-phosphorylated-β-Catenin, anti-GAPDH and anti-PCNA were obtained from Bioworld (USA); and FITC-labeled goat anti-rabbit IgG was from Sigma-Aldrich (USA). BSA was a product of Sigma-Aldrich (USA); bFGF was obtained from Proteintech (USA); EGF and B27 supplement were from Invitrogen (Thermo Fisher, USA), and ultra-low attachment plates were purchased from Corning Corp. (USA).
7
Co-immunoprecipitation and Immunoblotting Protocol
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For Co-immunoprecipitation (Co-IP) assays, 293T cells or LS174T cells were lysed with lysis buffer (150 mM NaCl, 200 mM Tris, 25 mM sodium pyrophosphate, 5 mM EDTA, 10 mM sodium orythovanadate, 10 mM glycerolphosphate, 50 mM NaF, 1mM PMSF and protein inhibitors cocktail). Total cell lysates were immunoprecipitated with anti-SRC-3 antibodies (Santa Cruz, C-20, sc-7216), anti-c-Fos antibodies (Santa Cruz, H-125, sc-7202) or control rabbit immunoglobulin G (IgG) (Sigma-Alrich, I5006). After washing five times with cell lysis buffer, samples were analyzed by immunoblot. For immunoblot analysis, LS174T cells were lysed in RAPA buffer (150 mM NaCl, 50 mM Tris, 0.1% SDS, 1 mM EDTA, 1% Triton X-100, 50 mM NaF, 1 mM PMSF and protease inhibitors cocktail). Proteins were quantified with BCA assay. Equal amount of proteins were loaded onto 8% sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore), followed by immunoblotting with anti-SRC-3 (cell signaling, 5E11, #2116), anti-KLF4 (Proteintech Group, 11880-1-AP), MUC2 (Santa Cruz, B306.1, sc-59859)or anti-β-actin (Sigma-Alrich, A5441). Western blots were analyzed by using a Tonen Image.
8
Western Blot Analysis of Key Markers
9
Protein Expression Analysis by Western Blot
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Cells seeded in 6-well plates were lysed by scraping into lysis buffer. The protein concentration of each lysate was examined using the BCA protein assay kit (TaKaRa, Japan). Western blotting was performed according to standard protocol with the following antibodies and dilutions: anti-KLF4 (Proteintech, #11880-1-AP, 1:1000), anti-CDH3 (Proteintech, #13773-1-AP, 1:500), anti-GSK-3β (Cell Signaling Technology, #12456, 1:1500), anti-Phospho-GSK-3β (Ser9) (Cell Signaling Technology, #9323, 1:1500), anti-AKT (Cell Signaling Technology, #9272, 1:1500), anti-Phospho-Akt (Ser473) (Cell Signaling Technology, #4051, 1:1500), anti-Erk1/2 (Thr202/Tyr204) (Cell Signaling Technology, #4695, 1:1500), anti-Phospho-Erk1/2 (Thr202/Tyr204) (Cell Signaling Technology, #4370, 1:1500), anti-p70S6K (Proteintech, #14485-1-AP, 1:500), anti-Phospho-p70 S6 Kinase (Cell Signaling Technology, #9204, 1:1500), anti-Smad3 (Cell Signaling Technology, #9513, 1:1500), anti-Phospho-Smad3 (Ser423/425) (Cell Signaling Technology, #9520, 1:1500), anti-β-catenin (Cell Signaling Technology, #8480, 1:1500) and anti-GAPDH (Proteintech, #60004-1-Ig, 1:10000).
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