Proteins from untransfected and stably transfected cell extracts were subjected to SDS-PAGE (12%) and blotted onto PVDF membranes (Immobilion, Millipore). Blots were then incubated with rabbit polyclonal antibody for SOD1 or SOD2 (Prestige, Sigma) and mouse monoclonal antibody for β-tubulin (Sigma). The PVDF membranes were probed with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Sigma). Immunoreactive proteins were visualized using enhanced chemiluminescence advance (ECL, GE Healthcare). Densitometry was carried out using Image J Software and the constitutively expressed β-tubulin protein was used as loading control.
Immobilion
Manufactured by Merck Group
Immobilion is a versatile laboratory equipment product developed by Merck Group. It is designed for the immobilization and purification of biomolecules, such as proteins, enzymes, and antibodies. The core function of Immobilion is to provide a reliable platform for researchers and scientists to isolate and concentrate target analytes from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Cellytic buffer, by Merck Group (3 mentions)
Nupage, by Thermo Fisher Scientific (3 mentions)
Chemilumi one chemiluminescent kit, by Nacalai Tesque (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Westernbreeze immunodetection system, by Thermo Fisher Scientific (2 mentions)
Mouse monoclonal antibody for β tubulin, by Merck Group (1 mentions)
Horseradish peroxidase conjugated anti rabbit or anti mouse igg, by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
Peroxidase linked protein a, by GE Healthcare (1 mentions)
Tgx gradient gel, by Bio-Rad (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Criterion xt, by Bio-Rad (1 mentions)
Chemodoc mp imaging system, by Bio-Rad (1 mentions)
Enhanced chemiluminescence, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Anti p27, by Santa Cruz Biotechnology (1 mentions)
11 protocols using immobilion
1
Western Blot Analysis of SOD1 and SOD2
2
Western Blot Analysis of CypA and SAMHD1
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Monocytes and macrophages (3.5×106 cells) were lysed in Laemmli sample buffer (100 mM Tris-HCl, pH 6.8, 0.04% sodium dodecyl sulfate (SDS), 20% glycerol, 0.12% 2-mercaptoethanol). Proteins in the lysates were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins in the gel were then electrically transferred to a membrane (Immobilion; Millipore, Billerica, MA). Blots were blocked and probed with anti-CypA affinity rabbit polyclonal antibody (Sigma, St. Louis, MO) overnight at 4°C. Blots then were incubated with peroxidase-linked protein A (GE Healthcare, Buckinghamshire, UK), and bound antibodies were visualized with a Chemilumi-One chemiluminescent kit (Nacalai Tesque, Kyoto, Japan). Quantities of cell lysate were normalized by CypA level, then subjected to a new round of SDS-PAGE and membrane transfer. For the new blot, SAMHD1 protein in the membrane was detected with anti-SAMHD1 (611–625) rabbit antibody (Sigma) followed by peroxidase-linked protein A (GE Healthcare, Buckinghamshire, UK) detection as described above.
3
Protein Extraction and Immunoblot Analysis
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Cells were lysed in RIPA buffer (Sigma) supplemented with 1% protease inhibitor ‘cocktail’ (Pierce). Proteins were then separated by electrophoresis through 4%–20% TGX gradient gels (BioRad) and were transferred to polyvinylidene fluoride membranes (Immobilion; Millipore). For the detection of human proteins by immunoblot analysis, we used primary antibodies listed in online supplemental table S5 and western enhanced chemiluminescence (BioRad). Blots were developed in ChemiDoc MP imaging system (BioRad).
4
Immunoblot Analysis of Protein Samples
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Cells were lysed in CelLytic buffer (Sigma) supplemented with 1% protease inhibitor ‘cocktail’ (Pierce). Protein samples were subsequently separated by electrophoresis through NuPAGE (Invitrogen) 4–12% Bis-Tris gradient gels or 4–12% pre-cast XT-Criterion gradient gels (Biorad, Hercules, CA), and were then transferred to PVDF membranes (Immobilion; Millipore). Afterwards, blots were blocked in 5% milk in TBST and incubated overnight at 4 °C with primary antibodies (Supplementary Table 4 ). After incubation with primary antibodies, blots were washed with 0.1% TBST three times for 10 minutes each, followed by incubation with secondary antibodies (goat anti-mouse or goat anti-rabbit antibodies, ThermoFisher Scientific) conjugated with alkaline phosphatase (AP) in 5% milk in TBST at room temperature for an hour. Washing was repeated as above, and blots were visualized using Novex™ AP Chemiluminescent Substrate (CDP-Star™) (ThermoFisher Scientific). Blots were developed either on films or on Biorad Chemodoc MP Imaging system with Image Lab 5.1 software.
5
Protein Extraction and Western Blot Analysis
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Total proteins were extracted from the cell lines using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China). Tissue lysates were centrifuged at 14,000 rpm for 5 min at 4°C, and the supernatants were collected for further analysis. The protein concentration was determined using BCA protein Assay Kit (Beyotime). Fifty μg of total protein were mixed with loading buffer, denatured at 95°C for 5 min, resolved on a 10% SDS polyacrylamide gels and electro-transferred to PVDF membranes (Bio-RAD, CA). After being blocked with 5% milk and 1% bovine serum albumin solution, the membrane was incubated at 4°C overnight with the following primary antibodies: anti-Cleaved-caspase3, anti-BCL-2, anti-Bax, anti-p-STAT5a, anti-STAT5a, anti-p-AKT, anti-AKT, anti-p-ERK1/2, anti-ERK1/2, and anti-GAPDH (Acbam, diluted 1:1000). On the following day, the membrane was washed 3 times with TBST and then incubated with horseradish peroxidase-conjugated secondary antibodies (Acbam, diluted 1:5,000) for 2 h at room temperature. The protein bands were developed with a chemiluminescence detection system (Immobilion, Millipore), and the relative band intensity was analyzed using ImageJ software.
6
Western Blot Protein Detection
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Cells were lysed in CelLytic buffer (Sigma, St. Louis, MO) supplemented with PMSF, phosphatase and protease inhibitor cocktail (Pierce, Rockford, IL). 15 μg of protein mixture per sample were separated on NuPAGE (Invitrogen, Carlsbad, CA) 4-12% Bis-Tris gradient gels and transferred on PVDF membranes (Immobilion, Millipore, Temecula, CA). For the detection of mouse and human proteins by Western blot, primary antibodies were used together with the WesternBreeze immunodetection system (Invitrogen). Details of antibodies used are in Supplementary Table 4 .
7
Quantifying PINK1 and Parkin Protein Levels
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To assess PINK1 and Parkin protein levels, transfected HEK293T and HeLa cells were lysed in 20 mm Tris–HCl buffer, pH 7.5, containing 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm beta-glycerophosphate, 1 mm Na3VO4, protease inhibitor cocktail (Sigma) and kept on ice for 30 min. Clarified lysates were obtained by centrifugation at 17 500g for 15 min at 4°C. The detergent-soluble supernatant fractions were quantified by BCA assay (Pierce Biotechnology). Proteins (50 µg) were separated by SDS-PAGE, transferred on PVDF membranes (Immobilion, Millipore) and subjected to western blot analysis using appropriate primary and secondary antibodies. Immunoreactive proteins were visualized using enhanced chemiluminescence (GE Healthcare). Densitometry was performed by using Image J Software and endogenous GAPDH protein was used as loading control.
8
Western Blot Analysis of Proteins
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Plasmid-transfected 293 T cells (6 × 105 cells) were lysed in 100 μL of lysis buffer (50 mM Tris–HCl at pH 7.5, 150 mM NaCl, 1% Nonidet P-40, and 0.5% sodium deoxycholate). Proteins in the lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proteins in the gel were then electrically transferred to a membrane (Immobilion; Millipore, Billerica, MA). Blots were blocked and probed with N2 mAb (HM1057, EastCoast Bio, Maryland Heights, MO) or C2 mAb (CV15, CerTest Biotec, Zaragoza, Spain) overnight at 4 °C. The blots were then incubated with peroxidase-linked anti-mouse IgG (H + L), and the bound antibodies were visualized with a Chemi-Lumi One chemiluminescent kit (Nacalai Tesque, Kyoto, Japan).
9
Western Blot Analysis of Cell Signaling
10
Protein Extraction and Western Blot Analysis
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Total proteins were extracted from the cell lines using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China). Tissue lysates were centrifuged at 14,000 rpm for 5 min at 4°C, and the supernatants were collected for further analysis. The protein concentration was determined using BCA protein Assay Kit (Beyotime). Fifty μg of total protein were mixed with loading buffer, denatured at 95°C for 5 min, resolved on a 10% SDS polyacrylamide gels and electro-transferred to PVDF membranes (Bio-RAD, CA). After being blocked with 5% milk and 1% bovine serum albumin solution, the membrane was incubated at 4°C overnight with the following primary antibodies: anti-Cleaved-caspase3, anti-BCL-2, anti-Bax, anti-p-STAT5a, anti-STAT5a, anti-p-AKT, anti-AKT, anti-p-ERK1/2, anti-ERK1/2, and anti-GAPDH (Acbam, diluted 1:1000). On the following day, the membrane was washed 3 times with TBST and then incubated with horseradish peroxidase-conjugated secondary antibodies (Acbam, diluted 1:5,000) for 2 h at room temperature. The protein bands were developed with a chemiluminescence detection system (Immobilion, Millipore), and the relative band intensity was analyzed using ImageJ software.
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