Cells were lysed in RIPA buffer (Solarbio) containing PMSF. Protein quantification was performed using the BCA Protein Quantification Kit (Solarbio). The protein sample was blended with the protein loading buffer and heated at 99°C for 5 minutes. The denatured proteins were separated by 10% SDS–PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). The PVDF membrane was placed in 5% skim milk for 1 hour and incubated at 4 ℃ overnight with the following primary antibodies: FOXP4 (1:1000, Abcam), GAPDH (1:10000, Abcam), MLL2 (1:1000, Abcam), E-cadherin (1:1000, Elabscience), vimentin (1:1000, Elabscience), and β-catenin (1:1000, Elabscience). Subsequently, the membranes were incubated with goat anti-rabbit IgG (KPL) for 1 hour, and then ECL reagent (Solarbio) was added for observation.
Foxp4
Manufactured by Abcam
Sourced in United States
FOXP4 is a transcription factor that belongs to the forkhead box (FOX) protein family. It plays a key role in the regulation of gene expression, cell differentiation, and development.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Gapdh, by Abcam (2 mentions)
Ecl reagent, by Solarbio (1 mentions)
β catenin, by Elabscience (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Vimentin, by Elabscience (1 mentions)
Bca protein quantification kit, by Solarbio (1 mentions)
Goat anti rabbit igg, by Solarbio (1 mentions)
E cadherin, by Elabscience (1 mentions)
Ripa lysate, by Beyotime (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Image lab software, by Cell Signaling Technology (1 mentions)
Clarity ecl chemiluminescent substrates, by Bio-Rad (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Ab17726, by Abcam (1 mentions)
Ab16645, by Abcam (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
4 protocols using foxp4
1
Protein Expression Analysis by Western Blot
2
FOXP4 Protein Expression Analysis
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Total proteins were extracted by lysing the cells with RIPA lysate (Beyotime, China). Then protein samples were subjected to SDS-PAGE and transferred to PVDF membranes (Millipore, USA). After blocking with 5% skim milk for 1 h at room temperature, the membranes were incubated with the primary antibody FOXP4 and GAPDH (Abcam, USA) at 4 °C for 12 h. Then the prepared membranes were incubated with secondary antibody for 2 h. Finally, the blots were detected by enhanced chemiluminescence kit (Pierce, Waltham, MA, USA).
3
Immunohistochemical Analysis of FOXP4 in THCA
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Thyroid cancer tissues and paired adjacent tissues were obtained from three patients with THCA in the Affiliated Hospital of Guizhou Medical University (Guizhou, China). The patients had not received any treatment before surgery. According to the microscopic examination results, the selected tissue paraffin sections should contain more tumor tissue and less interstitial components with avoidance of the necrotic and hemorrhagic areas. All specimens were formalin fixed and assayed according to the standard immunohistochemistry (IHC) protocols, and IHC staining was conducted following the manufacturer’s instructions. The adoptions of specific antibodies were as follows: FOXP4 (1:200, Abcam). FOXP4 expression was assessed based on the staining intensity (0, 1+, 2+, and 3+) and the percentage of positive cells. The scores of the expression were 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), or 4 (76–100%). The calculation of the staining index (SI) was as follows: SI = (the intensity score in 1) × (the intensity score in 2). Those with an SI < 3 were assigned as low expression and those with an SI ≥ 4 were assigned as high expression. In addition, the present study was approved by the Human Ethics Committee of the Affiliated Hospital of Guizhou Medical University.
4
Western Blot Analysis of FOXP1 and FOXP4
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Primary human neutrophils and HEK293 cells were solubilized in Lysis Buffer (20 mM Tris, pH 8, 150 mM NaCl, 1% Triton X-100, 3,5 mM sodium dodecyl sulfate, 13 mM deoxycholic acid) and extracted proteins were separated on a 4% to 15% Tris/Glycine/sodium dodecyl sulfate gel (Bio Rad). For transfer and blotting, we used a standard protocol. FOXP1 (#ab16645, abcam, 1:500) and FOXP4 (#ab17726, abcam, 1:500) primary antibodies were purchased from Abcam. The antirabbit IgG HRP-linked secondary antibody was purchased from Cell Signaling Technology and used at recommended concentrations. Signals of protein bands were detected using Clarity ECL chemiluminescent substrates (Bio-Rad) and ChemiDoc Imaging System (Bio-Rad). We quantified changes in protein levels relative to control using Image Lab software after normalization to β-actin or GAPDH (Cell Signaling Technologies), as indicated. Western blot images are representative of three biological replicates. Full western blots for FOXP1 and FOXP4 are provided in Figure S5
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