Human KLF4 Antibody

KLF4 in Human Colon.

KLF4 was detected in immersion fixed paraffin-embedded sections of human colon using 15 µg/mL Goat Anti-Human KLF4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3640) overnight at 4 °C. Tissue was stained with the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008) and counterstained with hematoxylin (blue). Specific labeling was localized to the nuclei of epithelial cells. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

Detection of Human KLF4 by Simple Western^TM.

Simple Western lane view shows lysates of HT-29 human colon adenocarcinoma cell line, loaded at 0.2 mg/mL. A specific band was detected for KLF4 at approximately 63 kDa (as indicated) using 25 µg/mL of Goat Anti-Human KLF4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3640) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system. Non-specific interaction with the 230 kDa Simple Western standard may be seen with this antibody.

Detection of KLF4 in HT-29 Human Colon Adenocarcinoma Cell Line (positive) and HDLM-2 Human Hodgkin’s Lymphoma Cell Line (negative) Cells.

KLF4 was detected in immersion fixed HT-29 Human Colon Adenocarcinoma Cell Line (positive) and HDLM-2 Human Hodgkin’s Lymphoma Cell Line (negative) Cells using Goat Anti-Human KLF4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3640) at 5 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red; Catalog # NL001) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

Detection of Mouse KLF4 by Knockdown Validated

KLF4 regulates the EndMT switch in CCM1 KO ECsA–DCultured lung‐derived WT and CCM1 KO ECs were lentivirally transduced with shRNA directed to either Klf4 (shKLF4) or control sequence (shCTRL). (A) qRT–PCR of mesenchymal (Fsp1, Id1), stem cell‐like (Sca1), and endothelial markers (VE‐cadherin and Claudin5) in WT shCTRL, WT shKLF4, CCM1 KO shCTRL, and CCM1 KO shKLF4 ECs. Data are mean ± SD (n = 3). Fold difference in gene expression is relative to WT shCTRL ECs. A two‐tailed unpaired t‐test was performed. Klf4: ****P < 0.00001, ***P = 0.0008, **P = 0.007; Fsp1: ***P = 0.0001, ****P = 3.9E‐05, ###P = 0.0002; Sca1: ***P = 0.0001, ****P < 0.00001; Id1: ***P = 0.0001; Ve‐cadherin: ***P = 0.0001; Claudin5: ****P = 1.37E‐05. (B) WB of EndMT markers in WT shCTRL, WT shKLF4, CCM1 KO shCTRL, and CCM1 KO shKLF4 ECs. Vinculin is the loading control. These data are representative of three independent observations. (C) Proliferation rate of WT shCTRL, WTshKLF4, CCM1 KO shCTRL, and CCM1 KO shKLF4 ECs cultured for 5 days. Columns represent mean ± SD (n = 8). A two‐tailed unpaired t‐test was performed. 3 days of culture: ***P = 0.0001, **P = 0.0045, ##P = 0.0017; 5 days of culture: ***P = 0.0001, **P = 0.0029, ##P = 0.0040. (D) Migration rate measured in a wound assay of WT shCTRL, CCM1 KO shCTRL, and CCM1 KO shKLF4 ECs. Mean ± SD is shown (n = 6). A two‐tailed unpaired t‐test was performed. ***P = 0.0004, ###P = 0.0009.ECultured lung‐derived WT ECs were lentivirally transduced with a full‐length murine Klf4 (LentiKLF4) or empty vector (Mock). qRT–PCR (left panel) and WB (right panel) of EndMT markers in Mock and LentiKLF4 ECs. qRT–PCR data are mean ± SD (n = 3) and the fold changes are relative to Mock ECs. A two‐tailed unpaired t‐test was performed. **P = 0.002, ***P = 0.0007, ###P = 0.0001. WB results are representative of three independent observations. Tubulin is the loading control.Source data are available online for this figure. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/26612856), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human KLF4 by Knockdown Validated

Klf4 significantly rescues the effects of HDAC1 on leukemia cell proliferation. (a) Effects of ectopic expression of Klf4 on HDAC1-induced cell proliferation. * and ** indicate P<0.05 and P<0.01, respectively. (b) FACS analysis showing the effects of ectopic expression of Klf4 on HDAC1-induced cell cycle. * and ** indicate P<0.05 and P<0.01, respectively. (c) CCK8 analysis showing the effects of Klf4 knockdown on HDAC1 deficiency-mediated cell proliferation. * and ** indicate P<0.05 and P<0.01, respectively. (d) FACS analysis showing effects of Klf4 knockdown on HDAC1 deficiency-mediated cell cycle. * and ** indicate P<0.05 and P<0.01, respectively. (e) Western blotting analyses showing the effects of ectopic expression of Klf4 and HDAC1 on the expression levels of p21 and p27. GAPDH was used as the loading control. (f) Western blotting analyses showing the effects Klf4 and HDAC1 knockdown on the expression levels of p21 and p27. GAPDH was used as the loading control. (g) ChIP-PCR assays showing Klf4 binding at the promoter region of p21 and p27 after HDAC1 knockdown. Control or HDAC1-deficient cells were fixed and subjected to ChIP assay with anti-Klf4 antibody. ChIP-PCR primers were used for p21 and p27 promoters where shown in Supplementary Table S2. *, **, and *** indicate P<0.05, P<0.01, and P<0.001, respectively Image collected and cropped by CiteAb from the following publication (https://www.nature.com/articles/cddis2014433), licensed under a CC-BY license. Not internally tested by R&D Systems.