Mouse KLF4 Antibody

Detection of KLF4-regulated Genes by Chromatin Immunoprecipitation. D3 mouse embryonic stem cell line was fixed using formaldehyde, resuspended in lysis buffer, and sonicated to shear chromatin. KLF4/DNA complexes were immunoprecipitated using 5 µg Goat Anti-Mouse KLF4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3158) or control antibody (AB-108-C) for 15 minutes in an ultrasonic bath, followed by Biotinylated Anti-Goat IgG Secondary Antibody (BAF109). Immunocomplexes were captured using 50 µL of MagCellect Streptavidin Ferrofluid (MAG999) and DNA was purified using chelating resin solution. TheB2Rpromoter was detected by standard PCR.

KLF4 in D3 Mouse Cell Line. KLF4 was detected in immersion fixed D3 mouse embryonic stem cell line using Mouse KLF4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3158) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red; NL001) and counterstained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

KLF4 in Mouse Colon Tissue. KLF4 was detected in perfusion fixed frozen sections of mouse colon tissue using Goat Anti-Mouse KLF4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3158) at 1 µg/mL overnight at 4 °C. Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (CTS013). Tissue was stained using the Anti-Goat IgG VisUCyte™ HRP Polymer Antibody (brown; VC004) and counterstained with hematoxylin (blue). Specific staining was localized to cell nuclei. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

Detection of Mouse KLF4 by Immunohistochemistry Overexpression of beta -catenin maintains B6 ESC self-renewal in the presence of LIF.(A) Immunoblot analysis of phospho-beta -catenin, beta -catenin and beta -actin levels in B6 ES cells cultured in 1000 U/ml LIF condition supplemented without or with 3 µM CH for 4 days. (B) Immunofluorescence analysis of 3 µM CH-treated and non-treated B6 ESC clones stained for beta -catenin and Hoechst for nuclei in the presence of LIF for 4 days. Scale bars represent 100 µm. (C) Quantitative RT-PCR analysis of Axin2, T and Cdx1 transcript levels in 3 µM CH-treated and control B6 ES cells. Error bars represent the SD of three biological replicates. (D) beta -catenin S33A protein stably expressed in ES cells was detected by immunoblotting. (E) Phase contrast microscopy image and alkaline phosphatase staining of control and beta -catenin S33A-transfected B6 ES cells in the presence of 1000 U/ml LIF for 4 days. Scale bars represent 100 µm. (F) Immunofluorescence staining for OCT4, NANOG, and KLF4 in mock and beta -catenin S33A-transfected B6 ES cells in the presence of 1000 U/ml LIF for 4 days. Cell nuclei were stained with Hoechst. Scale bars represent 100 µm. (G) Quantitative RT-PCR analysis of Oct4, Nanog, Rex1, Klf4, Tbx3, and Fgf5 transcript levels in mock and beta -catenin S33A-transfected B6 ES cells in the presence of 1000 U/ml LIF for 4 days. Error bars represent the SD of three biological replicates. (H) beta -catenin S33A delta C, beta -catenin S33A and TCF4 delta N protein were detected by immunoblotting with anti-flag tag antibody from extracts of ES cells transfected with PiggyBac plasmids. Quantitative RT-PCR analysis of Axin2, T and Cdx1 transcript levels in beta -catenin S33A delta C, beta -catenin S33A, TCF4 delta N, and mock-transfected B6 ES cells. Error bars represent the SD of three biological replicates. (I) Alkaline phosphatase staining of mock-transfected, and beta -catenin delta C over-expressing B6 ES cells cultured in LIF for 5 passages, and mock-transfected and TCF4 delta N-transfected B6 ESCs cultured in CHIR99021 plus LIF for 5 passages. Scale bars represent 100 µm. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0035892), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse KLF4 by Immunocytochemistry/Immunofluorescence LIF maintains B6 ES cell self-renewal in the presence of CHIR99021.(A) Phase contrast image of B6 ES cells in the presence of 1000 U/ml LIF supplemented without or with 3 µM CHIR99021 (CH) for 4 days. Scale bars represent 100 µm. (B) Alkaline phosphatase staining of B6 ES cells in the presence of 1000 U/ml LIF supplemented without or with 3 µM CH for 4 days. Scale bars represent 100 µm. (C) Immunofluorescence staining for OCT4, NANOG, and KLF4 in B6 ES cells treated with 1000 U/ml LIF and 3 µM CH for 4 days. Cell nuclei were stained using Hoechst. Scale bars represent 100 µm. (D) Relative quantification of Oct4, Nanog, Rex1, Klf4, Tbx3, and Fgf5 mRNA in B6 ES cells in 1000 U/ml LIF culture without or with 3 µM CH, by qRT-PCR. Error bars represent the SD of three biological replicates. (E) B6 ES cells,1×104/well, cultured in different concentrations of LIF, ranging from 0 to 10,000 U/ml, in the absence or presence of 3 µM CH for 5 days, were assessed for alkaline phosphatase activity. Scale bars represent 100 µm. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0035892), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse KLF4 by Chromatin Immunoprecipitation Overlapping SIM2 occupancy with master transcription factor binding sites.a. Frequency distribution of OCT4, SOX2 and NANOG DNA binding sites in a 40kb window centered to the newly identified SIM2 DNA binding sites. Plots show a significant enrichment for the OSN binding sites at the SIM2 peak localization in SIM2 A6 expressing cells. Pie charts show the proportion of SIM2 DNA binding sites overlapping with the OCT4, SOX2 or NANOG binding sites (in grey) (100bp window). p = Fisher’s exact test p-value; F score: measure of the significance of the association (1 = perfect match). b. Protein co-immunoprecipitation experiments of SIM2-FLAG with endogenous OCT4, SOX2, KLF4 (left panel) and NANOG (right panel). Cellular protein extracts from Sim2 expressing cells (A6) or EB3 cells were immunoprecipitated by using antibodies directed against each of the pluripotency factors (N-terminal and C-terminal part of NANOG) or IgG as a negative control for co-immunoprecipitation. Associated proteins were immunoblotted using an anti-FLAG antibody. Red star shows the SIM2-FLAG protein, blue star the signal given by the recognition of the IgG heavy chains. Ø: Beads only; kDa: kilodaltons; protein lysat: protein lysat was loaded as an input control for the immunoblot. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25955728), licensed under a CC-BY license. Not internally tested by R&D Systems.