Antibodies were purchased from the following companies: Mst1 (#3682; WB, 1:1000; IHC, 1:200), phospho-Mst1-Thr183 (#3681; WB, 1:1000; IHC, 1:200), phospho-Nur77-Ser351 (#5095; WB, 1:1000), Flag (#14793; WB, 1:1000), Myc (#2276; WB, 1:1000; IF, 1:100) (all from Cell Signalling Technology), phosphothreonine (#P6623; WB, 1:500), phosphoserine (#P5747; WB, 1:1000) (all from Millipore Sigma), Nur77 (#ab48789, Abcam; WB, 1:1000; IHC, 1:200), HOXA10 (#sc-17158; WB, 1:1000), and phospho-Nur77-Ser341 (#sc-16991; WB, 1:1000) (all from Santa Cruz Biotechnology, Inc.), β3-integrin (#BS3660; WB, 1:1000), GAPDH (#AP0063; WB, 1:2000) (all from Bioworld Technology, Inc.), FAK (#CY5464; WB, 1:1000), phospho-FKA-Y397 (#CY5464; WB, 1:1000) (all from Abways Technology, Inc.). The phosphorylation state-specific antibody against phospho-Nur77-Thr366 was provided by Nanjing Peptide Biotech (WB, 1:400). As brief, the synthetic phosphorylated peptide ANLLTpSLVRA was subjected to immunization of rabbits, following with a two-step affinity purification with a non-phosphorylated peptide column and a phosphorylated peptide column to get the polyclonal antibody against phospho-Nur77-Thr366.
Nur77
Manufactured by Abcam
Sourced in United Kingdom, United States
Nur77 is a nuclear receptor that functions as a transcriptional regulator. It is involved in the regulation of various cellular processes, including cell proliferation, apoptosis, and metabolism. Nur77 is expressed in a variety of tissues and cell types, and its activity is modulated by various signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Abcam (2 mentions)
Hoxa10, by Santa Cruz Biotechnology (1 mentions)
Flag tag (flag), by Cell Signaling Technology (1 mentions)
Phosphoserine, by Merck Group (1 mentions)
Gapdh, by Bioworld Technology (1 mentions)
Enhanced chemiluminescence substrate, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Huabio (1 mentions)
Gapdh, by Huabio (1 mentions)
Immobilon p transfer membrane, by Merck Group (1 mentions)
Bca protein assay, by Merck Group (1 mentions)
Ecl kit, by Merck Group (1 mentions)
C jun, by Abcam (1 mentions)
P c jun, by Abcam (1 mentions)
3β hsd, by Santa Cruz Biotechnology (1 mentions)
P450scc, by Cell Signaling Technology (1 mentions)
Sr b1, by Abcam (1 mentions)
C fos, by Bioworld Technology (1 mentions)
17β hsd, by Santa Cruz Biotechnology (1 mentions)
β tubulin, by Cell Signaling Technology (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
4 protocols using nur77
1
Phosphorylation-specific Antibody Generation
2
Western Blot Analysis of Nur77 Expression
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Equal amounts of extracts (30 μg) were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (10%) and transferred onto polyvinylidene fluoride (0.45 μm) membranes, followed by blocking with 5% skimmed milk at room temperature for 1 h. Subsequently, the membranes were incubated with the primary antibodies Nur77 (1:2000 dilution; Abcam) and GAPDH (1:5000 dilution; Huabio) at 4°C overnight. After washing five times with TBS‐T, the membranes were incubated with horseradish peroxidase‐conjugated secondary antibody (1:50,000 dilution; Huabio) for 1 h. Western blot bands were visualized using enhanced chemiluminescence substrate (Millipore) and analyzed by Image J software.
3
Western Blot Analysis of Nur77 and GFP
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Cells were lysed in 150 mM NaCl, 10 mM Tris (pH 7.5), 5 mM EDTA, 1% Triton X-100, 1 mM PMSF, 10 mg/ml leupeptin, 10 mg/ml pepstatin, and 10 mg/ml aprotinin for 30 min on ice. Equal amounts of lysates (50 μg) were separated by 8–12% SDS-PAGE and transferred onto Immobilon-P transfer membranes (Millipore, Billerica, MA). Non-specific binding sites were blocked by incubating membranes in 5% (w/v) solution of nonfat dried milk in TBST (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 0.1% Tween 20). Blocked membranes were probed with specific antibodies against Nur77 (1;1000, Abcam, Cambridge, UK), GFP (1;1000, eBioscience, San Diego, CA), or β-actin (1;5000, BioVision, Mountain View, CA) in TBST. Membranes were washed 3 times with TBST and then incubated for 2 h at room temperature in TBST containing HRP-linked anti-rabbit or anti-mouse immunoglobulin. After 3 washes in TBST, HRP was visualized by chemiluminescence using the ECL kit (Chemicon, Temecula, CA). The protein concentration was quantified using the BCA protein assay (Merck, Darmstadt, Germany).
4
Protein Extraction and Western Blot Analysis
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Proteins were extracted from rat testes and TM3 cells using Cell Lysis Buffer for Western blot and IP. Concentrations of protein were determined using BCA protein assay kits (Beyotime Biotech Inc., China). Twenty μg of protein were separated and electrophoresed by SDS-PAGE. Nitrocellulose membranes were blocked by TBST containing 5% non-fat milk for 1 h at 37 °C, followed by incubations with specific primary antibodies overnight at 4 °C and HRP-conjugated secondary antibodies for 1 h at 37 °C. Target proteins were visualized and analyzed using the ECL system. ERK, p-ERK, JNK, p-JNK, p38, p-p38, P450scc, GAPDH, and β-Tubulin antibodies were bought from the Cell Signaling Technology Inc. (USA). Map3k1, SRD5A2, Nur77, c-Jun, p-c-Jun, SRB1, StAR, P450c17, and β-Actin antibodies were gotten from the Abcam Inc. (USA). AR, ERα, SF-1, 3β-HSD, and 17β-HSD antibodies were purchased from the Santa Cruz Biotechnology Inc. (USA). SRD5A1, c-Fos, and p-c-Fos antibodies were obtained from the Bioworld Technology Inc. (China).
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