Mouse KLF4 Antibody

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 deltaC, beta-catenin S33A and TCF4 deltaN 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 deltaC, beta-catenin S33A, TCF4 deltaN, 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 deltaC over-expressing B6 ES cells cultured in LIF for 5 passages, and mock-transfected and TCF4 deltaN-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.