These procedures were performed as described [63 (link)]. To cross-link proteins, cells were incubated with 1 mM DSP (dithiobis[succinimidylpropionate]) (Thermo Fisher Scientific) at room temperature for 30 min. Antibodies against UCHL1 (R&D Systems, Inc.), Lamin A/C (Upstate Biotechnology), HDAC1 (Millipore), actin (Sigma-Aldrich), GAPDH (Santa Cruz Biotechnology), p53 (Abcam), TRF1 (Novus Biologicals), TRF2 (Novus Biologicals), RAP1 (TERF2IP) (Bethyl Laboratories) and KSRP (a gift from Dr. Douglas L. Black) were used.
Uchl1
Manufactured by R&D Systems
Sourced in United States
UCHL1 is a deubiquitinating enzyme that plays a role in the regulation of protein ubiquitination. It is involved in the processing of ubiquitin precursors and ubiquitinated proteins.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Protease and phosphatase inhibitors cocktail, by Merck Group (2 mentions)
Psma7, by Thermo Fisher Scientific (2 mentions)
Actin hrp, by Merck Group (2 mentions)
Uchl1, by Cell Signaling Technology (2 mentions)
Hdac1, by Merck Group (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
S6 ribosomal protein, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by R&D Systems (1 mentions)
4e bp1, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Glial fibrillary acidic protein (gfap), by R&D Systems (1 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Cd4 pacific blue, by BioLegend (1 mentions)
Defa1, by R&D Systems (1 mentions)
Anti sheep igg nl557, by R&D Systems (1 mentions)
Foxp3 transcription factor staining kit, by Thermo Fisher Scientific (1 mentions)
Cd45ro fitc, by BioLegend (1 mentions)
Cd45ra apc, by BioLegend (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
5 protocols using uchl1
1
Cross-Linking Proteins in Cells
2
Quantitative Immunoblotting of Cellular Proteins
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Immunoblotting was performed using a standard protocol as described earlier [35] . Cells were lysed in NP-40 buffer containing protease and phosphatase inhibitors cocktails (Millipore), 0.2mM phenylmethylsulfonyl fluoride, and 10mM N' ethylmalamide. Protein quantification was conducted using the Pierce BCA protein assay kit (Thermo Fisher #23225). Proteins were resolved by 4-20% gradient SDS-PAGE. Primary antibodies used were UCHL1 (13179, Cell Signaling), UCHL1 (MAB6007, R&D Systems), PSMA7 (cat# PA5-22289; Invitrogen), ATF3 (cat# 33593; Cell signaling), APEH (cat# 376612; Santacruz Biotechnology), and actin-HRP (Sigma).
3
Western Blot Analysis of Cell Signaling
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Immunoblotting was performed using a standard protocol as described previously (35) . Cells were lysed in NP-40 buffer containing Protease and Phosphatase Inhibitors Cocktails (Millipore), 0.2 mmol/L phenylmethylsulfonyl fluoride, and 10 mmol/L N 0 ethylmalamide. Protein quantification was conducted using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, #23225). Proteins were resolved by 4%-20% gradient SDS-PAGE. Primary antibodies were procured from Cell Signaling Technology: UCHL1 (13179), ATF3 (33593), ATF4 (11845), pSer51 eIF2a (3398), cleaved caspase-3 (9664), pSer235/236 S6 Ribosomal Protein (4858), S6 Ribosomal Protein (2217), 4E-BP1 (9452), and pThr37/46 4E-BP1 (2855). Other antibodies included UCHL1 (MAB6007, R&D Systems), PSMA7 (PA5-22289, Invitrogen), APEH (376612, Santa Cruz Biotechnology), cleaved caspase-3 (AF835, R&D Systems), and actin-HRP (Sigma).
4
Biomarker Analysis of CSF Samples
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CSF samples were collected at the time of diagnosis, and directly transported to the Centre of Clinical Laboratory, where the samples were centrifuged. The supernatants of the CSF samples were collected and stored at -80 °C until assayed ( Lin et al. , 2016 ) . CSF UCH-L1 (R&D Systems, Minneapolis, MN, USA), GFAP (R&D Systems, Minneapolis, MN, USA), and NF-L (Novus Biologicals, Littleton, USA) levels were measured using human enzyme-linked immunosorbent assay (ELISA) kits, according to the manufacturer's instructions.
5
Viability and Surface Antigen Analysis
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Cells were harvested after stimulation and viability of cells post activation was measured using the LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific). Surface staining of antigen was performed at 4 C for 20 minutes. For intracellular antigens, cells were fixed and permeabilized using the FOXP3/Transcription factor staining kit (eBioscience), and incubated with the appropriate antibody for 1 hour at 4 C. Acquisition of cells was performed using the BD FACSCantoTM II Flow Cytometer and subsequent analysis was performed using FlowJo (v9 or v10).
The clone and dilution of antibodies used for flow cytometry were as follows: CD4-Pacific Blue (Biolegend, OKT4, 1:200), CD69-APC (Biolegend, FN50, 1:200), CD45RA-APC (Biolegend, HI100, 1:200), CD45RO-FITC (Biolegend, UCHL1, 1:200), DEFA1 (R&D Systems, 2 g/ml), anti-sheep IgG-NL557 (R&D Systems, 1:500). IFN expression was analyzed using the IFN-Secretion Assay (PE, Miltenyi Biotec) following manufacturers instruction.
The clone and dilution of antibodies used for flow cytometry were as follows: CD4-Pacific Blue (Biolegend, OKT4, 1:200), CD69-APC (Biolegend, FN50, 1:200), CD45RA-APC (Biolegend, HI100, 1:200), CD45RO-FITC (Biolegend, UCHL1, 1:200), DEFA1 (R&D Systems, 2 g/ml), anti-sheep IgG-NL557 (R&D Systems, 1:500). IFN expression was analyzed using the IFN-Secretion Assay (PE, Miltenyi Biotec) following manufacturers instruction.
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