The analyzed cells were lysed as previously described.3 (link) Subsequently, the collected lysate was centrifuged at 4°C at 18,624xg for 15 min and protein concentration was determined by use of the Bradford method. In total, 50 µg protein was loaded and separated by SDS-PAGE and transferred onto Immobilon-P membranes using the iBlot apparatus (Invitrogen; Thermo Fisher Scientific, Inc.). After blocking in 5% fat-free milk, the membrane was incubated with the appropriate diluted primary antibody overnight at 4°C. The primary antibodies were used with the following dilution: Actin (Transgen Biotech Co., Ltd.; 1:5,000), ZNF750 (Abcam; 1:500) and KDR (Abcam; 1:200). Antibody binding was detected with horseradish-peroxidase-conjugated anti-mouse (Sigma-Aldrich; Merck KGaA) or anti-rabbit (Cell Signaling Technology, Inc.) antibodies for 1 h at room temperature and chemiluminescence was measured using a LAS4000 Device Chemiluminescence System (Beijing Sage Creation Science Co., Ltd.). Each experiment was repeated at least 3 times.
Znf750
Manufactured by Abcam
ZNF750 is a zinc finger protein that functions as a transcriptional regulator. It contains a C2H2-type zinc finger domain and is involved in the regulation of gene expression.
Automatically generated - may contain errors
2 protocols using znf750
1
Western Blot Analysis of Protein Lysates
2
ChIP Assay of Transcription Factors
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ChIP assays were performed using Pierce Agarose ChIP kit (Thermo Scientific, prod#26156)
Following the manufacture’s protocol. Briefly, human primary KC were differentiated by elevated the calcium to 1.3mM and cultured for 3 days. The cells were then incubated with 1% formaldehyde for 10 minutes at 37°C, followed by incubation with 1x Glycine solution for 5 minutes in room temperature. The cells were then collected, lysed and digested by MNase. Protein-chromatin complexes were immunoprecipitated with antibodies against KLF4, ZNF750, or rabbit IgG (negative control). Anti-KLF4, ZNF750 and rabbit IgG were purchased from Abcam (Cambridge, MA). Complexes were separated by incubation with ChIP grade proteinA/G agarose. Chromatin DNA fragments were then eluted and purified for quantitative real-time PCR. The primers used for amplification of specific regions of FOXC1 promoter were described in supplemental materials.
Following the manufacture’s protocol. Briefly, human primary KC were differentiated by elevated the calcium to 1.3mM and cultured for 3 days. The cells were then incubated with 1% formaldehyde for 10 minutes at 37°C, followed by incubation with 1x Glycine solution for 5 minutes in room temperature. The cells were then collected, lysed and digested by MNase. Protein-chromatin complexes were immunoprecipitated with antibodies against KLF4, ZNF750, or rabbit IgG (negative control). Anti-KLF4, ZNF750 and rabbit IgG were purchased from Abcam (Cambridge, MA). Complexes were separated by incubation with ChIP grade proteinA/G agarose. Chromatin DNA fragments were then eluted and purified for quantitative real-time PCR. The primers used for amplification of specific regions of FOXC1 promoter were described in supplemental materials.
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