Protein identification by immunoblotting was performed following an optimized protocol as previously described [2 (link)]. Antibodies were obtained from the following sources: anti-p-STAT5 and JAM-A (Abcam, Cambridge, MA, USA) and anti-STAT5, MX1 and ß-actin from Cell Signaling (Danvers, MA, USA).
Anti p stat5
Manufactured by Abcam
Sourced in United States
Anti-p-STAT5 is a primary antibody that recognizes the phosphorylated form of the STAT5 transcription factor. STAT5 is a key mediator of cytokine signaling and plays a role in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one system, by Bio-Rad (2 mentions)
Anti stat5, by Abcam (2 mentions)
Anti socs1, by Abcam (2 mentions)
Anti stat5, by Cell Signaling Technology (1 mentions)
Jam a, by Abcam (1 mentions)
Corresponding secondary antibody, by Abcam (1 mentions)
Anti jak1, by Abcam (1 mentions)
Anti caveolin 1, by Abcam (1 mentions)
Anti caspase 3, by Abcam (1 mentions)
Anti pp2a, by Abcam (1 mentions)
Anti pias1, by Abcam (1 mentions)
Anti pias3, by Abcam (1 mentions)
Anti jak3, by Abcam (1 mentions)
Anti pim1, by Abcam (1 mentions)
Anti bcl 2, by Abcam (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Anti bax, by Abcam (1 mentions)
Anti cd4, by Abcam (1 mentions)
Streptavidin peroxidase ihc assay kit, by ZSGB-BIO (1 mentions)
Anti il 17a, by Santa Cruz Biotechnology (1 mentions)
4 protocols using anti p stat5
1
Immunoblotting for Protein Identification
2
Quantification of Colon Tissue Proteins
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Colon tissues (n = 6) were quickly shredded and mixed with protease inhibitor cocktails, which were randomly selected from every group. The mixture was homogenized by ultrasound on crushed ice and centrifuged at 13000g at 4°C for 10 min; the supernatant was extracted; and the protein concentration was determined by BCA protein assay. Equal amounts of total protein (60 μg) were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane using a semidry transfer slot (Bio-Rad, USA). After blocking with 5% nonfat dried milk for 1 h, the membrane was incubated with proportionally diluted antibodies at 4°C overnight. The antibodies included rabbit anti-GAPDH (1:2,000), anti-BCL-2 (1:2,000), anti-BIM-1 (1:1,000), anti-caspase-3 (1:500), anti-BAX (1:1,000), anti-PP2A (1:1,000), anti-β-casein (1:2,500), anti-caveolin-1 (1:1,000), anti-Pim-1 (1:1,000), anti-JAK1 (1:1,000), anti-JAK3 (1:1,000), anti-PIAS1 (1:2,000), anti-PIAS3 (1:2,000), anti-Socs-1 (1:1,000), anti-P-STAT5 (1:1,000), and anti-STAT5 (1:1,000; Abcam). Then, samples were incubated with a corresponding secondary antibody (Abcam) for 1 h. After washing three times with TBST, the blots were visualized by the Proteogel imaging system (FluorChem M, ProteinSimple, USA) and quantified with the Quantity One System (Bio-Rad).
3
Immunohistochemical Analysis of pSS Tissues
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Human MSG tissues from 10 pSS patients and 7 Sicca control patients were obtained after informed consent. The gland tissues were fixed with 4% paraformaldehyde, followed with embedding in paraffin blocks. The slides were heated at 65°C for 30 min, followed by paraffin removal with xylene and subsequent rehydration with ethanol. Antigen retrieval was performed in citrate buffer (pH 6.0) at a sub-boiling temperature for 20 min. Samples were blocked with 10% goat serum for 1 h at RT and incubated with primary antibody (1:100) overnight at 4°C. The slides were visualized using streptavidin peroxidase IHC assay kit (ZSGB-bio, China) and counter stained with hematoxylin. The primary antibody of immunohistochemistry was performed using anti-IL-17A (Clone G-4, Santa Cruz), anti-CD4 (Clone EPR6855, Abcam), anti-p-STAT3 (Clone EP2147Y, Abcam)m and anti-p-STAT5 (Clone E208, Abcam). Images were obtained using an OLYMPUS-BX51 microscope at 10 × 10 or 40 × 10 magnification.
4
Colon Tissue Protein Extraction and Analysis
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Colon tissues (n = 6) were quickly shredded and mixed with protease inhibitor cocktails. The mixture was homogenized by ultrasound on crushed ice and centrifuged at 13000 g at 4°C for 10 min, the supernatant was extracted, and the protein concentration was determined by BCA protein assay. Equal amounts of total protein (60 µg) were separated by SDS-PAGE and transferred to polyvinylidene uoride (PVDF) membrane using a semi-dry transfer slot (BIO-RAD, USA). After blocking with 5% nonfat dried milk for 1 h, the membrane was incubated with proportionally diluted antibodies at 4°C overnight. The antibodies included rabbit anti-GAPDH (1:2000), anti-BCL-2 (1:2000), anti-BIM-1 (1:1000), anti-Caspase-3 (1:500), anti-BAX (1:1000), anti-PP2A (1:1000), anti-β-casein (1:2500), anti-Caveolin-1 (1:1000), anti-Pim-1 (1:1000), anti-JAK1 (1:1000), anti-JAK3 (1:1000), anti-PIAS1 (1:2000), anti-PIAS3 (1:2000), anti-Socs-1
(1:1000), anti-P-STAT5 (1:1000), and anti-STAT5 (1:1000)(Abcam). Then, samples were incubated with a corresponding secondary antibody (Abcam) for 1 h. After washing three times with TBST, the blots were visualized by the Proteogel imaging system (FluorChem M, ProteinSimple, USA) and quanti ed with the Quantity One System (Bio-Rad).
(1:1000), anti-P-STAT5 (1:1000), and anti-STAT5 (1:1000)(Abcam). Then, samples were incubated with a corresponding secondary antibody (Abcam) for 1 h. After washing three times with TBST, the blots were visualized by the Proteogel imaging system (FluorChem M, ProteinSimple, USA) and quanti ed with the Quantity One System (Bio-Rad).
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