Structured Review

PerkinElmer s l methionine
S L Methionine, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/s l methionine/product/PerkinElmer
Average 94 stars, based on 6 article reviews
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s l methionine - by Bioz Stars, 2020-02
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Filtration:

Article Title: Homeostatic adaptation to endoplasmic reticulum stress depends on Ire1 kinase activity
Article Snippet: Cells were grown to an OD600 of 0.3, harvested by filtration, and resuspended in media lacking methionine. .. At the time of UPR induction, 1 µCi/ml [35 S]methionine (PerkinElmer) plus 50 µM cold methionine was added to cells.

Positive Control:

Article Title: Identification of Unique Antigenic Determinants in the Amino Terminus of IA-2 (ICA512) in Childhood and Adult Autoimmune Diabetes: New Biomarker Development
Article Snippet: IA-2ec was in vitro transcribed/translated in the presence of [35 S]methionine (PerkinElmer) using the TNT-coupled rabbit reticulocyte system (Promega, Madison, WI) with T7 RNA polymerase. .. We used the protein tyrosine phosphatase IA-2 Q-20 antibody (sc-54749; Santa Cruz Biotechnology, Santa Cruz, CA) as positive control and serum from individuals without diabetes as negative controls.

Synthesized:

Article Title: MEHMO syndrome mutation EIF2S3-I259M impairs initiator Met-tRNAiMet binding to eukaryotic translation initiation factor eIF2
Article Snippet: .. Aminoacylation reactions with 5 μM synthesized tRNAi Met , 2 mM adenosine triphosphate-Mg2+ , 0.3 μM [35 S]methionine (Perkin Elmer), 10 mM MgCl2 and 1 μM MetRS were incubated in 1× Reaction Buffer (100 mM HEPES-KOH (pH 7.5), 1 mM DTT and 10 mM KCl) for 30 min at 30°C, and aminoacylated Met-tRNAi Met was purified by phenol/chloroform extraction ( ). .. For measurements of tRNA binding, seven 2-fold serial dilutions of eIF2 with final concentrations from 3.125 to 200 nM were incubated for 15 min at 26°C in Binding Assay Buffer (25 mM HEPES-KOH (pH 7.6), 2.5 mM Mg(OAc)2 , 80 mM KOAc (pH 7.6), 2 mM DTT and 0.5 mM guanosine 5′-[β,γ-imido]triphosphate (GDPNP)-Mg2+ ).

Article Title: Cap-Independent Translation Promotes C. elegans Germ Cell Apoptosis through Apaf-1/CED-4 in a Caspase-Dependent Mechanism
Article Snippet: Protein substrates were labeled with [35 S]-methionine (Perkin Elmer Life Sciences) using TnT T3 coupled reticulocyte lysate system (Promega). .. One microliter of in vitro synthesized product was incubated with 10 µl of recombinant CED-3 at 37°C for 2 h. Control reactions substituted with 10 µl of non-catalytic BSA were prepared in CED-3 reaction buffer.

Article Title: Opaque-2 Regulates a Complex Gene Network Associated with Cell Differentiation and Storage Functions of Maize Endosperm [OPEN]
Article Snippet: .. Using the resulting constructs as templates, tagged proteins were synthesized in vitro with [35 S]methionine (Perkin-Elmer) using the TnT Quick Coupled Transcription/Translation Systems (Promega) following the manufacturer’s instructions. .. The in vitro pull-down assays were performed as previously described ( ) with minor modifications.

Autoradiography:

Article Title: Oxidized DJ-1 Interacts with the Mitochondrial Protein BCL-XL
Article Snippet: .. The cells were then labeled with 200 μCi/ml [35 S]methionine (PerkinElmer) for 2 h. Pulse-labeling was terminated by incubating cells in fresh medium containing an excess of unlabeled methionine (300 mg/liter), and the cells were harvested at 0, 7, and 14 h. Cells lysates were immunoprecipitated with anti-GFP antibodies (Santa Cruz Biotechnology) and analyzed by SDS-PAGE and autoradiography. .. Western blot densitometric analysis of three independent experiments was performed using Photoshop 7.0 (Adobe).

Article Title: METTL3 promotes translation in human cancer cells
Article Snippet: Cells were then incubated for 20 min after supplementation with [35 S]-methionine ([35 S]-Met; 100 mCi/mL; PerkinElmer), washed twice with ice-cold PBS, harvested, and lysed. .. For the quantitation of [35 S]-Met-labeled proteins, cell lysates were subjected to 4–12% Tris-Glycine SDS-PAGE and analyzed by autoradiography.

Article Title: Nuclear Aggresomes Form by Fusion of PML-associated Aggregates V⃞
Article Snippet: COS-7 cells were transfected with either GFP170* or GFP-250 construct in a six-well plate for 24 h. Cells were then washed in PBS and incubated in methionine-free DMEM for 1 h. Cells were labeled with 200 μCi/ml [35 S]methionine (PerkinElmer Life and Analytical Sciences, Boston, MA) for 60 min. Incorporation was terminated by washing the cells with PBS and chasing with DMEM medium supplemented with 0.2 mM methionine for indicated times. .. Equal amounts of lysate from each time point were resolved by 10% SDS-PAGE followed by autoradiography using a PhosphoImage screen.

Article Title: Negative Regulation of Prolactin Receptor Stability and Signaling Mediated by SCFβ-TrCP E3 Ubiquitin Ligase
Article Snippet: Ubiquitination reactions were carried out with a total volume of 20 μl containing 2 μg of ubiquitin, 20 pmol of E1, and 100 pmol of E2 as well as 2 mM ATP at 37°C for 60 min. Boiling in SDS-sample buffer terminated the reactions, and the products were analyzed by SDS-PAGE and autoradiography. .. After starvation in DMEM lacking methionine and cysteine and metabolic labeling with a [35 S]methionine-[35 S]cysteine mixture (Perkin-Elmer), cells were harvested at each chase time point with complete DMEM supplemented with FBS (0.5%), PRL (20 ng/ml), and unlabeled methionine and cysteine (2 mM).

Construct:

Article Title: Nuclear Aggresomes Form by Fusion of PML-associated Aggregates V⃞
Article Snippet: .. COS-7 cells were transfected with either GFP170* or GFP-250 construct in a six-well plate for 24 h. Cells were then washed in PBS and incubated in methionine-free DMEM for 1 h. Cells were labeled with 200 μCi/ml [35 S]methionine (PerkinElmer Life and Analytical Sciences, Boston, MA) for 60 min. Incorporation was terminated by washing the cells with PBS and chasing with DMEM medium supplemented with 0.2 mM methionine for indicated times. .. At each time point, cells were lysed with RIPA buffer supplemented with protease inhibitor cocktail and 1.0 mM PMSF.

Article Title: Identification of Unique Antigenic Determinants in the Amino Terminus of IA-2 (ICA512) in Childhood and Adult Autoimmune Diabetes: New Biomarker Development
Article Snippet: IA-2 Autoantibody Radiobinding Assays The IA-2ec construct (amino acids 26–577) contains the entire extracellular domain minus the signal peptide ( , ). .. IA-2ec was in vitro transcribed/translated in the presence of [35 S]methionine (PerkinElmer) using the TNT-coupled rabbit reticulocyte system (Promega, Madison, WI) with T7 RNA polymerase.

Article Title: Opaque-2 Regulates a Complex Gene Network Associated with Cell Differentiation and Storage Functions of Maize Endosperm [OPEN]
Article Snippet: .. Using the resulting constructs as templates, tagged proteins were synthesized in vitro with [35 S]methionine (Perkin-Elmer) using the TnT Quick Coupled Transcription/Translation Systems (Promega) following the manufacturer’s instructions. .. The in vitro pull-down assays were performed as previously described ( ) with minor modifications.

Ubiquitin Assay:

Article Title: Negative Regulation of Prolactin Receptor Stability and Signaling Mediated by SCFβ-TrCP E3 Ubiquitin Ligase
Article Snippet: For the in vitro ubiquitination assay, GST-PRLR proteins were expressed in bacteria, purified by using glutathione beads, and preincubated (1 μg) with 25-μg extracts from 293T cells treated with PRL (200 ng/ml for 20 min) in a kinase buffer containing 50 mM Tris HCl (pH 7.5), 5 mM MgCl2 , 0.5 mM dithiothreitol, 5 mM NaF, 10 nM okadaic acid, 50 μM ATP, and 10 μM [32 P-γ]ATP for 30 min at 30°C. .. After starvation in DMEM lacking methionine and cysteine and metabolic labeling with a [35 S]methionine-[35 S]cysteine mixture (Perkin-Elmer), cells were harvested at each chase time point with complete DMEM supplemented with FBS (0.5%), PRL (20 ng/ml), and unlabeled methionine and cysteine (2 mM).

Quantitation Assay:

Article Title: METTL3 promotes translation in human cancer cells
Article Snippet: Cells were then incubated for 20 min after supplementation with [35 S]-methionine ([35 S]-Met; 100 mCi/mL; PerkinElmer), washed twice with ice-cold PBS, harvested, and lysed. .. For the quantitation of [35 S]-Met-labeled proteins, cell lysates were subjected to 4–12% Tris-Glycine SDS-PAGE and analyzed by autoradiography.

Expressing:

Article Title: Oxidized DJ-1 Interacts with the Mitochondrial Protein BCL-XL
Article Snippet: H1299 cells stably expressing EGFP-Bcl-XL were starved in methionine-free medium and incubated for 30 min. .. The cells were then labeled with 200 μCi/ml [35 S]methionine (PerkinElmer) for 2 h. Pulse-labeling was terminated by incubating cells in fresh medium containing an excess of unlabeled methionine (300 mg/liter), and the cells were harvested at 0, 7, and 14 h. Cells lysates were immunoprecipitated with anti-GFP antibodies (Santa Cruz Biotechnology) and analyzed by SDS-PAGE and autoradiography.

Article Title: MEHMO syndrome mutation EIF2S3-I259M impairs initiator Met-tRNAiMet binding to eukaryotic translation initiation factor eIF2
Article Snippet: His-tagged MetRS was purified from Escherichia coli XL1 Blue cells (Agilent) carrying the E. coli methionyl-tRNA synthetase (MetRS) expression plasmid pRA101 ( ) as described previously ( ). .. Aminoacylation reactions with 5 μM synthesized tRNAi Met , 2 mM adenosine triphosphate-Mg2+ , 0.3 μM [35 S]methionine (Perkin Elmer), 10 mM MgCl2 and 1 μM MetRS were incubated in 1× Reaction Buffer (100 mM HEPES-KOH (pH 7.5), 1 mM DTT and 10 mM KCl) for 30 min at 30°C, and aminoacylated Met-tRNAi Met was purified by phenol/chloroform extraction ( ).

Article Title: The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers
Article Snippet: A plasmid encoding the capsid and 3C protease genome regions of the O1 Manisa strain of FMDV in pBG200 T7 expression vectors ( ) were used as a template to generate the plasmids pBG200-P1-2A and pBG200-P1-Δ2A, with the primer sequences described in , by PCR. .. Reaction mixtures contained 20 ng/μl plasmid DNA, 8 Bq/μl [35 S]methionine (EasyTag; PerkinElmer), and various concentrations of hsp90 inhibitor and were incubated at 30°C for 1.5 h. Processing and assembly of P1-2A into pentamers was achieved by the addition of 1 μM 3Cpro to the reaction and incubation at 37°C for 1 h. Following incubation, free radiolabel was removed from reactions by dialysis at 4°C against phosphate-buffered saline (PBS), using mini-dialysis units (Slide-A-Lyzer; Pierce) that had been preblocked with 1% bovine serum albumin in PBS.

BIA-KA:

Article Title: Granzyme B Inhibits Vaccinia Virus Production through Proteolytic Cleavage of Eukaryotic Initiation Factor 4 Gamma 3
Article Snippet: Evaluation of protein synthesis Jurkat cells were pulse-labeled with 33 µCi [35 S]methionine (PerkinElmer) for 1 hour at a density of 3×105 cells/ml. .. Evaluation of protein synthesis Jurkat cells were pulse-labeled with 33 µCi [35 S]methionine (PerkinElmer) for 1 hour at a density of 3×105 cells/ml.

Transfection:

Article Title: Nuclear Aggresomes Form by Fusion of PML-associated Aggregates V⃞
Article Snippet: .. COS-7 cells were transfected with either GFP170* or GFP-250 construct in a six-well plate for 24 h. Cells were then washed in PBS and incubated in methionine-free DMEM for 1 h. Cells were labeled with 200 μCi/ml [35 S]methionine (PerkinElmer Life and Analytical Sciences, Boston, MA) for 60 min. Incorporation was terminated by washing the cells with PBS and chasing with DMEM medium supplemented with 0.2 mM methionine for indicated times. .. At each time point, cells were lysed with RIPA buffer supplemented with protease inhibitor cocktail and 1.0 mM PMSF.

Article Title: Negative Regulation of Prolactin Receptor Stability and Signaling Mediated by SCFβ-TrCP E3 Ubiquitin Ligase
Article Snippet: Briefly, cells were grown in 100-mm-diameter dishes and transfected with the indicated plasmids. .. After starvation in DMEM lacking methionine and cysteine and metabolic labeling with a [35 S]methionine-[35 S]cysteine mixture (Perkin-Elmer), cells were harvested at each chase time point with complete DMEM supplemented with FBS (0.5%), PRL (20 ng/ml), and unlabeled methionine and cysteine (2 mM).

TCA Precipitation:

Article Title: Homeostatic adaptation to endoplasmic reticulum stress depends on Ire1 kinase activity
Article Snippet: At the time of UPR induction, 1 µCi/ml [35 S]methionine (PerkinElmer) plus 50 µM cold methionine was added to cells. .. Cells were lysed, total protein was isolated by TCA precipitation, and scintillation counts were measured.

Stable Transfection:

Article Title: Oxidized DJ-1 Interacts with the Mitochondrial Protein BCL-XL
Article Snippet: H1299 cells stably expressing EGFP-Bcl-XL were starved in methionine-free medium and incubated for 30 min. .. The cells were then labeled with 200 μCi/ml [35 S]methionine (PerkinElmer) for 2 h. Pulse-labeling was terminated by incubating cells in fresh medium containing an excess of unlabeled methionine (300 mg/liter), and the cells were harvested at 0, 7, and 14 h. Cells lysates were immunoprecipitated with anti-GFP antibodies (Santa Cruz Biotechnology) and analyzed by SDS-PAGE and autoradiography.

Pulse Chase:

Article Title: Oxidized DJ-1 Interacts with the Mitochondrial Protein BCL-XL
Article Snippet: Paragraph title: Pulse Chase Analysis ... The cells were then labeled with 200 μCi/ml [35 S]methionine (PerkinElmer) for 2 h. Pulse-labeling was terminated by incubating cells in fresh medium containing an excess of unlabeled methionine (300 mg/liter), and the cells were harvested at 0, 7, and 14 h. Cells lysates were immunoprecipitated with anti-GFP antibodies (Santa Cruz Biotechnology) and analyzed by SDS-PAGE and autoradiography.

Article Title: Absence of the Yeast Hsp31 Chaperones of the DJ-1 Superfamily Perturbs Cytoplasmic Protein Quality Control in Late Growth Phase
Article Snippet: Paragraph title: Pulse-chase analysis ... After 50 min of starvation cells were labelled with 0.2 mCi of [35 S]-methionine (0.37 MBq/μl; PerkinElmer Life Sciences) for 20 min. Chase medium containing 40 mM non-radioactive methionine was then added to the cells and samples were collected at defined time points.

Article Title: Negative Regulation of Prolactin Receptor Stability and Signaling Mediated by SCFβ-TrCP E3 Ubiquitin Ligase
Article Snippet: Pulse-chase analysis was carried out with 293T cells as described elsewhere ( ). .. After starvation in DMEM lacking methionine and cysteine and metabolic labeling with a [35 S]methionine-[35 S]cysteine mixture (Perkin-Elmer), cells were harvested at each chase time point with complete DMEM supplemented with FBS (0.5%), PRL (20 ng/ml), and unlabeled methionine and cysteine (2 mM).

Activation Assay:

Article Title: Proteolytic degradation and potential role of onconeural protein cdr2 in neurodegeneration
Article Snippet: In vitro and cell-based calpain cleavage assay For in vitro calpain cleavage assays, the vector encoding T7-tagged mouse cdr2 in pcDNA3 was transcribed and translated in the presence of [35 S]-methionine (Perkin Elmer, Boston, MA, USA) using a TnT Quick coupled transcription/translation system (Promega, Madison, WI, USA) according to the manufacturer's recommendations. .. [35 S]-cdr2 or cell lysates (50 μ g) were incubated for 1 h at 30 °C in a calpain activation buffer containing 1 mM CaCl2 in the presence or absence of purified m-calpain (0.343 units) or μ -calpain (0.134 units; both from Calbiochem) as recommended by the manufacturer.

Protease Inhibitor:

Article Title: Proteolytic degradation and potential role of onconeural protein cdr2 in neurodegeneration
Article Snippet: In vitro and cell-based calpain cleavage assay For in vitro calpain cleavage assays, the vector encoding T7-tagged mouse cdr2 in pcDNA3 was transcribed and translated in the presence of [35 S]-methionine (Perkin Elmer, Boston, MA, USA) using a TnT Quick coupled transcription/translation system (Promega, Madison, WI, USA) according to the manufacturer's recommendations. .. For cell-based cleavage assay, MN9D cells were lysed in buffer containing 50 mM Tris-HCl, pH 8.0, 2 mM EDTA, and 1% Triton X-100 buffer without protease inhibitor cocktail.

Article Title: Nuclear Aggresomes Form by Fusion of PML-associated Aggregates V⃞
Article Snippet: COS-7 cells were transfected with either GFP170* or GFP-250 construct in a six-well plate for 24 h. Cells were then washed in PBS and incubated in methionine-free DMEM for 1 h. Cells were labeled with 200 μCi/ml [35 S]methionine (PerkinElmer Life and Analytical Sciences, Boston, MA) for 60 min. Incorporation was terminated by washing the cells with PBS and chasing with DMEM medium supplemented with 0.2 mM methionine for indicated times. .. At each time point, cells were lysed with RIPA buffer supplemented with protease inhibitor cocktail and 1.0 mM PMSF.

Cell Culture:

Article Title: mTOR-Mediated Activation of p70 S6K Induces Differentiation of Pluripotent Human Embryonic Stem Cells
Article Snippet: .. H7 hESC and H7 Diff were cultured as described above in the presence of 10 μCi/mL [35 S]methionine (Perkin-Elmer, Waltham, MA, USA). ..

Generated:

Article Title: The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers
Article Snippet: Radiolabeled P1-2A was generated from plasmids using in vitro transcription and translation reactions in rabbit reticulocyte lysates (TnT quick; Promega) according to the manufacturer's instructions. .. Reaction mixtures contained 20 ng/μl plasmid DNA, 8 Bq/μl [35 S]methionine (EasyTag; PerkinElmer), and various concentrations of hsp90 inhibitor and were incubated at 30°C for 1.5 h. Processing and assembly of P1-2A into pentamers was achieved by the addition of 1 μM 3Cpro to the reaction and incubation at 37°C for 1 h. Following incubation, free radiolabel was removed from reactions by dialysis at 4°C against phosphate-buffered saline (PBS), using mini-dialysis units (Slide-A-Lyzer; Pierce) that had been preblocked with 1% bovine serum albumin in PBS.

Protein Concentration:

Article Title: Granzyme B Inhibits Vaccinia Virus Production through Proteolytic Cleavage of Eukaryotic Initiation Factor 4 Gamma 3
Article Snippet: Evaluation of protein synthesis Jurkat cells were pulse-labeled with 33 µCi [35 S]methionine (PerkinElmer) for 1 hour at a density of 3×105 cells/ml. .. Evaluation of protein synthesis Jurkat cells were pulse-labeled with 33 µCi [35 S]methionine (PerkinElmer) for 1 hour at a density of 3×105 cells/ml.

Sequencing:

Article Title: The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers
Article Snippet: PCRs were performed using KOD polymerase (Roche), and a VP0 G2A substitution to prevent myristoylation was introduced into the coding sequence of pBG200-P1-Δ2A to generate pBG200-P1-2A-G2A using QuikChange lightning mutagenesis (Agilent) and the primer sequences described in . .. Reaction mixtures contained 20 ng/μl plasmid DNA, 8 Bq/μl [35 S]methionine (EasyTag; PerkinElmer), and various concentrations of hsp90 inhibitor and were incubated at 30°C for 1.5 h. Processing and assembly of P1-2A into pentamers was achieved by the addition of 1 μM 3Cpro to the reaction and incubation at 37°C for 1 h. Following incubation, free radiolabel was removed from reactions by dialysis at 4°C against phosphate-buffered saline (PBS), using mini-dialysis units (Slide-A-Lyzer; Pierce) that had been preblocked with 1% bovine serum albumin in PBS.

Recombinant:

Article Title: Cap-Independent Translation Promotes C. elegans Germ Cell Apoptosis through Apaf-1/CED-4 in a Caspase-Dependent Mechanism
Article Snippet: rCasp-3/rCED-3 cleavage assays and immunoblotting Recombinant enzymes were pre-incubated at 37°C for 30 min to 1 h prior to all cleavage assays. .. Protein substrates were labeled with [35 S]-methionine (Perkin Elmer Life Sciences) using TnT T3 coupled reticulocyte lysate system (Promega).

Cleavage Assay:

Article Title: Proteolytic degradation and potential role of onconeural protein cdr2 in neurodegeneration
Article Snippet: .. In vitro and cell-based calpain cleavage assay For in vitro calpain cleavage assays, the vector encoding T7-tagged mouse cdr2 in pcDNA3 was transcribed and translated in the presence of [35 S]-methionine (Perkin Elmer, Boston, MA, USA) using a TnT Quick coupled transcription/translation system (Promega, Madison, WI, USA) according to the manufacturer's recommendations. .. For cell-based cleavage assay, MN9D cells were lysed in buffer containing 50 mM Tris-HCl, pH 8.0, 2 mM EDTA, and 1% Triton X-100 buffer without protease inhibitor cocktail.

Radioactivity:

Article Title: Functional Importance of Dicer Protein in the Adaptive Cellular Response to Hypoxia *
Article Snippet: Radioactivity from each well was counted in a liquid scintillation counter. .. As a complementary approach, [35 S]methionine (PerkinElmer Life Sciences) was added to the growth medium of confluent HUVEC grown on 12-well plates at a final concentration of 10 mCi/well for the last 0, 20, 40, or 60 min of normoxia or hypoxia after which cells were washed with ice-cold PBS, incubated in 10% TCA for 20 min, washed three times with 100% ethanol, and dried at 45 °C.

Pull Down Assay:

Article Title: Opaque-2 Regulates a Complex Gene Network Associated with Cell Differentiation and Storage Functions of Maize Endosperm [OPEN]
Article Snippet: Paragraph title: In Vitro Pull-Down Assay ... Using the resulting constructs as templates, tagged proteins were synthesized in vitro with [35 S]methionine (Perkin-Elmer) using the TnT Quick Coupled Transcription/Translation Systems (Promega) following the manufacturer’s instructions.

Mutagenesis:

Article Title: The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers
Article Snippet: PCRs were performed using KOD polymerase (Roche), and a VP0 G2A substitution to prevent myristoylation was introduced into the coding sequence of pBG200-P1-Δ2A to generate pBG200-P1-2A-G2A using QuikChange lightning mutagenesis (Agilent) and the primer sequences described in . .. Reaction mixtures contained 20 ng/μl plasmid DNA, 8 Bq/μl [35 S]methionine (EasyTag; PerkinElmer), and various concentrations of hsp90 inhibitor and were incubated at 30°C for 1.5 h. Processing and assembly of P1-2A into pentamers was achieved by the addition of 1 μM 3Cpro to the reaction and incubation at 37°C for 1 h. Following incubation, free radiolabel was removed from reactions by dialysis at 4°C against phosphate-buffered saline (PBS), using mini-dialysis units (Slide-A-Lyzer; Pierce) that had been preblocked with 1% bovine serum albumin in PBS.

Isolation:

Article Title: Homeostatic adaptation to endoplasmic reticulum stress depends on Ire1 kinase activity
Article Snippet: At the time of UPR induction, 1 µCi/ml [35 S]methionine (PerkinElmer) plus 50 µM cold methionine was added to cells. .. Cells were lysed, total protein was isolated by TCA precipitation, and scintillation counts were measured.

Article Title: Negative Regulation of Prolactin Receptor Stability and Signaling Mediated by SCFβ-TrCP E3 Ubiquitin Ligase
Article Snippet: Proteins were captured on SCFβ-TrCP isolated from transfected 293T cells with HA antibody (Roche) and protein A beads (Invitrogen) as previously described ( ) and washed with buffer containing 300 mM NaCl, 0.5% Nonidet P-40, and phosphatase inhibitors. .. After starvation in DMEM lacking methionine and cysteine and metabolic labeling with a [35 S]methionine-[35 S]cysteine mixture (Perkin-Elmer), cells were harvested at each chase time point with complete DMEM supplemented with FBS (0.5%), PRL (20 ng/ml), and unlabeled methionine and cysteine (2 mM).

Labeling:

Article Title: Oxidized DJ-1 Interacts with the Mitochondrial Protein BCL-XL
Article Snippet: .. The cells were then labeled with 200 μCi/ml [35 S]methionine (PerkinElmer) for 2 h. Pulse-labeling was terminated by incubating cells in fresh medium containing an excess of unlabeled methionine (300 mg/liter), and the cells were harvested at 0, 7, and 14 h. Cells lysates were immunoprecipitated with anti-GFP antibodies (Santa Cruz Biotechnology) and analyzed by SDS-PAGE and autoradiography. .. Western blot densitometric analysis of three independent experiments was performed using Photoshop 7.0 (Adobe).

Article Title: METTL3 promotes translation in human cancer cells
Article Snippet: Paragraph title: Metabolic labeling ... Cells were then incubated for 20 min after supplementation with [35 S]-methionine ([35 S]-Met; 100 mCi/mL; PerkinElmer), washed twice with ice-cold PBS, harvested, and lysed.

Article Title: Cap-Independent Translation Promotes C. elegans Germ Cell Apoptosis through Apaf-1/CED-4 in a Caspase-Dependent Mechanism
Article Snippet: .. Protein substrates were labeled with [35 S]-methionine (Perkin Elmer Life Sciences) using TnT T3 coupled reticulocyte lysate system (Promega). .. Ten microliter reactions were prepared containing 0.5 µg of plasmid DNA and 0.8 µl of [35 S]methionine (10 mCi/mL).

Article Title: Nuclear Aggresomes Form by Fusion of PML-associated Aggregates V⃞
Article Snippet: .. COS-7 cells were transfected with either GFP170* or GFP-250 construct in a six-well plate for 24 h. Cells were then washed in PBS and incubated in methionine-free DMEM for 1 h. Cells were labeled with 200 μCi/ml [35 S]methionine (PerkinElmer Life and Analytical Sciences, Boston, MA) for 60 min. Incorporation was terminated by washing the cells with PBS and chasing with DMEM medium supplemented with 0.2 mM methionine for indicated times. .. At each time point, cells were lysed with RIPA buffer supplemented with protease inhibitor cocktail and 1.0 mM PMSF.

Article Title: Identification of Unique Antigenic Determinants in the Amino Terminus of IA-2 (ICA512) in Childhood and Adult Autoimmune Diabetes: New Biomarker Development
Article Snippet: IA-2ec was in vitro transcribed/translated in the presence of [35 S]methionine (PerkinElmer) using the TNT-coupled rabbit reticulocyte system (Promega, Madison, WI) with T7 RNA polymerase. .. IA-2ec autoantibodies were detected by radiobinding assay using 50% protein A–Sepharose to separate free [35 S]methionine from antibody-bound labeled products.

Article Title: Negative Regulation of Prolactin Receptor Stability and Signaling Mediated by SCFβ-TrCP E3 Ubiquitin Ligase
Article Snippet: .. After starvation in DMEM lacking methionine and cysteine and metabolic labeling with a [35 S]methionine-[35 S]cysteine mixture (Perkin-Elmer), cells were harvested at each chase time point with complete DMEM supplemented with FBS (0.5%), PRL (20 ng/ml), and unlabeled methionine and cysteine (2 mM). .. Harvested cells were lysed, and PRLR proteins were immunoprecipitated with Flag or PRLR antibody, separated on SDS-PAGE gel, and analyzed by autoradiography.

Purification:

Article Title: MEHMO syndrome mutation EIF2S3-I259M impairs initiator Met-tRNAiMet binding to eukaryotic translation initiation factor eIF2
Article Snippet: .. Aminoacylation reactions with 5 μM synthesized tRNAi Met , 2 mM adenosine triphosphate-Mg2+ , 0.3 μM [35 S]methionine (Perkin Elmer), 10 mM MgCl2 and 1 μM MetRS were incubated in 1× Reaction Buffer (100 mM HEPES-KOH (pH 7.5), 1 mM DTT and 10 mM KCl) for 30 min at 30°C, and aminoacylated Met-tRNAi Met was purified by phenol/chloroform extraction ( ). .. For measurements of tRNA binding, seven 2-fold serial dilutions of eIF2 with final concentrations from 3.125 to 200 nM were incubated for 15 min at 26°C in Binding Assay Buffer (25 mM HEPES-KOH (pH 7.6), 2.5 mM Mg(OAc)2 , 80 mM KOAc (pH 7.6), 2 mM DTT and 0.5 mM guanosine 5′-[β,γ-imido]triphosphate (GDPNP)-Mg2+ ).

Article Title: The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers
Article Snippet: The Δ2A versions of P1 were designed to encode the first 12 bases of the 2A protein-coding region followed by a His6 tag to allow purification in other experiments. .. Reaction mixtures contained 20 ng/μl plasmid DNA, 8 Bq/μl [35 S]methionine (EasyTag; PerkinElmer), and various concentrations of hsp90 inhibitor and were incubated at 30°C for 1.5 h. Processing and assembly of P1-2A into pentamers was achieved by the addition of 1 μM 3Cpro to the reaction and incubation at 37°C for 1 h. Following incubation, free radiolabel was removed from reactions by dialysis at 4°C against phosphate-buffered saline (PBS), using mini-dialysis units (Slide-A-Lyzer; Pierce) that had been preblocked with 1% bovine serum albumin in PBS.

Article Title: Proteolytic degradation and potential role of onconeural protein cdr2 in neurodegeneration
Article Snippet: In vitro and cell-based calpain cleavage assay For in vitro calpain cleavage assays, the vector encoding T7-tagged mouse cdr2 in pcDNA3 was transcribed and translated in the presence of [35 S]-methionine (Perkin Elmer, Boston, MA, USA) using a TnT Quick coupled transcription/translation system (Promega, Madison, WI, USA) according to the manufacturer's recommendations. .. [35 S]-cdr2 or cell lysates (50 μ g) were incubated for 1 h at 30 °C in a calpain activation buffer containing 1 mM CaCl2 in the presence or absence of purified m-calpain (0.343 units) or μ -calpain (0.134 units; both from Calbiochem) as recommended by the manufacturer.

Article Title: Granzyme B Inhibits Vaccinia Virus Production through Proteolytic Cleavage of Eukaryotic Initiation Factor 4 Gamma 3
Article Snippet: Evaluation of protein synthesis Jurkat cells were pulse-labeled with 33 µCi [35 S]methionine (PerkinElmer) for 1 hour at a density of 3×105 cells/ml. .. Following treatment, cells were centrifuged for 1 min at 18,000 g. Proteins were recovered through acetone purification using the ReadyPrep 2-D Cleanup kit.

Article Title: Negative Regulation of Prolactin Receptor Stability and Signaling Mediated by SCFβ-TrCP E3 Ubiquitin Ligase
Article Snippet: For the in vitro ubiquitination assay, GST-PRLR proteins were expressed in bacteria, purified by using glutathione beads, and preincubated (1 μg) with 25-μg extracts from 293T cells treated with PRL (200 ng/ml for 20 min) in a kinase buffer containing 50 mM Tris HCl (pH 7.5), 5 mM MgCl2 , 0.5 mM dithiothreitol, 5 mM NaF, 10 nM okadaic acid, 50 μM ATP, and 10 μM [32 P-γ]ATP for 30 min at 30°C. .. After starvation in DMEM lacking methionine and cysteine and metabolic labeling with a [35 S]methionine-[35 S]cysteine mixture (Perkin-Elmer), cells were harvested at each chase time point with complete DMEM supplemented with FBS (0.5%), PRL (20 ng/ml), and unlabeled methionine and cysteine (2 mM).

Polymerase Chain Reaction:

Article Title: The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers
Article Snippet: A plasmid encoding the capsid and 3C protease genome regions of the O1 Manisa strain of FMDV in pBG200 T7 expression vectors ( ) were used as a template to generate the plasmids pBG200-P1-2A and pBG200-P1-Δ2A, with the primer sequences described in , by PCR. .. Reaction mixtures contained 20 ng/μl plasmid DNA, 8 Bq/μl [35 S]methionine (EasyTag; PerkinElmer), and various concentrations of hsp90 inhibitor and were incubated at 30°C for 1.5 h. Processing and assembly of P1-2A into pentamers was achieved by the addition of 1 μM 3Cpro to the reaction and incubation at 37°C for 1 h. Following incubation, free radiolabel was removed from reactions by dialysis at 4°C against phosphate-buffered saline (PBS), using mini-dialysis units (Slide-A-Lyzer; Pierce) that had been preblocked with 1% bovine serum albumin in PBS.

Immunoprecipitation:

Article Title: Oxidized DJ-1 Interacts with the Mitochondrial Protein BCL-XL
Article Snippet: .. The cells were then labeled with 200 μCi/ml [35 S]methionine (PerkinElmer) for 2 h. Pulse-labeling was terminated by incubating cells in fresh medium containing an excess of unlabeled methionine (300 mg/liter), and the cells were harvested at 0, 7, and 14 h. Cells lysates were immunoprecipitated with anti-GFP antibodies (Santa Cruz Biotechnology) and analyzed by SDS-PAGE and autoradiography. .. Western blot densitometric analysis of three independent experiments was performed using Photoshop 7.0 (Adobe).

Article Title: Negative Regulation of Prolactin Receptor Stability and Signaling Mediated by SCFβ-TrCP E3 Ubiquitin Ligase
Article Snippet: After starvation in DMEM lacking methionine and cysteine and metabolic labeling with a [35 S]methionine-[35 S]cysteine mixture (Perkin-Elmer), cells were harvested at each chase time point with complete DMEM supplemented with FBS (0.5%), PRL (20 ng/ml), and unlabeled methionine and cysteine (2 mM). .. Harvested cells were lysed, and PRLR proteins were immunoprecipitated with Flag or PRLR antibody, separated on SDS-PAGE gel, and analyzed by autoradiography.

Blocking Assay:

Article Title: Opaque-2 Regulates a Complex Gene Network Associated with Cell Differentiation and Storage Functions of Maize Endosperm [OPEN]
Article Snippet: Using the resulting constructs as templates, tagged proteins were synthesized in vitro with [35 S]methionine (Perkin-Elmer) using the TnT Quick Coupled Transcription/Translation Systems (Promega) following the manufacturer’s instructions. .. Subsequently, 300 μL of blocking buffer (buffer W-100 [20 mM TrisOAc, pH 7.5, 10% glycerol, 1 mM EDTA, 5 mM MgCl2 , 0.2 M NaCl, 1% Nonidet P-40, 0.5 mM sodium deoxycholate, and 100 mM potassium glutamate] containing 0.5 mg/mL BSA, 0.5 mg/mL lysozyme, 0.05 mg/mL glycogen, and 1 mM DTT) was added and the mixture was incubated at 4°C for 1 h while rotating.

Staining:

Article Title: METTL3 promotes translation in human cancer cells
Article Snippet: Cells were then incubated for 20 min after supplementation with [35 S]-methionine ([35 S]-Met; 100 mCi/mL; PerkinElmer), washed twice with ice-cold PBS, harvested, and lysed. .. Colloidal blue staining was performed to verify equal amounts of protein loaded.

IA:

Article Title: Identification of Unique Antigenic Determinants in the Amino Terminus of IA-2 (ICA512) in Childhood and Adult Autoimmune Diabetes: New Biomarker Development
Article Snippet: .. IA-2ec was in vitro transcribed/translated in the presence of [35 S]methionine (PerkinElmer) using the TNT-coupled rabbit reticulocyte system (Promega, Madison, WI) with T7 RNA polymerase. .. IA-2ec autoantibodies were detected by radiobinding assay using 50% protein A–Sepharose to separate free [35 S]methionine from antibody-bound labeled products.

SDS Page:

Article Title: Oxidized DJ-1 Interacts with the Mitochondrial Protein BCL-XL
Article Snippet: .. The cells were then labeled with 200 μCi/ml [35 S]methionine (PerkinElmer) for 2 h. Pulse-labeling was terminated by incubating cells in fresh medium containing an excess of unlabeled methionine (300 mg/liter), and the cells were harvested at 0, 7, and 14 h. Cells lysates were immunoprecipitated with anti-GFP antibodies (Santa Cruz Biotechnology) and analyzed by SDS-PAGE and autoradiography. .. Western blot densitometric analysis of three independent experiments was performed using Photoshop 7.0 (Adobe).

Article Title: METTL3 promotes translation in human cancer cells
Article Snippet: Cells were then incubated for 20 min after supplementation with [35 S]-methionine ([35 S]-Met; 100 mCi/mL; PerkinElmer), washed twice with ice-cold PBS, harvested, and lysed. .. For the quantitation of [35 S]-Met-labeled proteins, cell lysates were subjected to 4–12% Tris-Glycine SDS-PAGE and analyzed by autoradiography.

Article Title: Nuclear Aggresomes Form by Fusion of PML-associated Aggregates V⃞
Article Snippet: COS-7 cells were transfected with either GFP170* or GFP-250 construct in a six-well plate for 24 h. Cells were then washed in PBS and incubated in methionine-free DMEM for 1 h. Cells were labeled with 200 μCi/ml [35 S]methionine (PerkinElmer Life and Analytical Sciences, Boston, MA) for 60 min. Incorporation was terminated by washing the cells with PBS and chasing with DMEM medium supplemented with 0.2 mM methionine for indicated times. .. Equal amounts of lysate from each time point were resolved by 10% SDS-PAGE followed by autoradiography using a PhosphoImage screen.

Article Title: Negative Regulation of Prolactin Receptor Stability and Signaling Mediated by SCFβ-TrCP E3 Ubiquitin Ligase
Article Snippet: Ubiquitination reactions were carried out with a total volume of 20 μl containing 2 μg of ubiquitin, 20 pmol of E1, and 100 pmol of E2 as well as 2 mM ATP at 37°C for 60 min. Boiling in SDS-sample buffer terminated the reactions, and the products were analyzed by SDS-PAGE and autoradiography. .. After starvation in DMEM lacking methionine and cysteine and metabolic labeling with a [35 S]methionine-[35 S]cysteine mixture (Perkin-Elmer), cells were harvested at each chase time point with complete DMEM supplemented with FBS (0.5%), PRL (20 ng/ml), and unlabeled methionine and cysteine (2 mM).

Plasmid Preparation:

Article Title: MEHMO syndrome mutation EIF2S3-I259M impairs initiator Met-tRNAiMet binding to eukaryotic translation initiation factor eIF2
Article Snippet: His-tagged MetRS was purified from Escherichia coli XL1 Blue cells (Agilent) carrying the E. coli methionyl-tRNA synthetase (MetRS) expression plasmid pRA101 ( ) as described previously ( ). .. Aminoacylation reactions with 5 μM synthesized tRNAi Met , 2 mM adenosine triphosphate-Mg2+ , 0.3 μM [35 S]methionine (Perkin Elmer), 10 mM MgCl2 and 1 μM MetRS were incubated in 1× Reaction Buffer (100 mM HEPES-KOH (pH 7.5), 1 mM DTT and 10 mM KCl) for 30 min at 30°C, and aminoacylated Met-tRNAi Met was purified by phenol/chloroform extraction ( ).

Article Title: The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers
Article Snippet: .. Reaction mixtures contained 20 ng/μl plasmid DNA, 8 Bq/μl [35 S]methionine (EasyTag; PerkinElmer), and various concentrations of hsp90 inhibitor and were incubated at 30°C for 1.5 h. Processing and assembly of P1-2A into pentamers was achieved by the addition of 1 μM 3Cpro to the reaction and incubation at 37°C for 1 h. Following incubation, free radiolabel was removed from reactions by dialysis at 4°C against phosphate-buffered saline (PBS), using mini-dialysis units (Slide-A-Lyzer; Pierce) that had been preblocked with 1% bovine serum albumin in PBS. ..

Article Title: Proteolytic degradation and potential role of onconeural protein cdr2 in neurodegeneration
Article Snippet: .. In vitro and cell-based calpain cleavage assay For in vitro calpain cleavage assays, the vector encoding T7-tagged mouse cdr2 in pcDNA3 was transcribed and translated in the presence of [35 S]-methionine (Perkin Elmer, Boston, MA, USA) using a TnT Quick coupled transcription/translation system (Promega, Madison, WI, USA) according to the manufacturer's recommendations. .. For cell-based cleavage assay, MN9D cells were lysed in buffer containing 50 mM Tris-HCl, pH 8.0, 2 mM EDTA, and 1% Triton X-100 buffer without protease inhibitor cocktail.

Article Title: Cap-Independent Translation Promotes C. elegans Germ Cell Apoptosis through Apaf-1/CED-4 in a Caspase-Dependent Mechanism
Article Snippet: Protein substrates were labeled with [35 S]-methionine (Perkin Elmer Life Sciences) using TnT T3 coupled reticulocyte lysate system (Promega). .. Ten microliter reactions were prepared containing 0.5 µg of plasmid DNA and 0.8 µl of [35 S]methionine (10 mCi/mL).

Software:

Article Title: Nuclear Aggresomes Form by Fusion of PML-associated Aggregates V⃞
Article Snippet: COS-7 cells were transfected with either GFP170* or GFP-250 construct in a six-well plate for 24 h. Cells were then washed in PBS and incubated in methionine-free DMEM for 1 h. Cells were labeled with 200 μCi/ml [35 S]methionine (PerkinElmer Life and Analytical Sciences, Boston, MA) for 60 min. Incorporation was terminated by washing the cells with PBS and chasing with DMEM medium supplemented with 0.2 mM methionine for indicated times. .. The relative radioactive intensity of GFP170* and GFP-250 bands was quantified and compared using ImageQuant software.

Negative Control:

Article Title: Identification of Unique Antigenic Determinants in the Amino Terminus of IA-2 (ICA512) in Childhood and Adult Autoimmune Diabetes: New Biomarker Development
Article Snippet: IA-2ec was in vitro transcribed/translated in the presence of [35 S]methionine (PerkinElmer) using the TNT-coupled rabbit reticulocyte system (Promega, Madison, WI) with T7 RNA polymerase. .. The assay was run in triplicate, and the results were expressed as an index calculated as follows: index = (serum sample counts per minute [cpm] − negative control cpm)/(positive control cpm − negative control cpm).

In Vitro:

Article Title: MEHMO syndrome mutation EIF2S3-I259M impairs initiator Met-tRNAiMet binding to eukaryotic translation initiation factor eIF2
Article Snippet: Yeast tRNAi Met was synthesized by in vitro transcription with T7 polymerase as described previously ( ). .. Aminoacylation reactions with 5 μM synthesized tRNAi Met , 2 mM adenosine triphosphate-Mg2+ , 0.3 μM [35 S]methionine (Perkin Elmer), 10 mM MgCl2 and 1 μM MetRS were incubated in 1× Reaction Buffer (100 mM HEPES-KOH (pH 7.5), 1 mM DTT and 10 mM KCl) for 30 min at 30°C, and aminoacylated Met-tRNAi Met was purified by phenol/chloroform extraction ( ).

Article Title: The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers
Article Snippet: Radiolabeled P1-2A was generated from plasmids using in vitro transcription and translation reactions in rabbit reticulocyte lysates (TnT quick; Promega) according to the manufacturer's instructions. .. Reaction mixtures contained 20 ng/μl plasmid DNA, 8 Bq/μl [35 S]methionine (EasyTag; PerkinElmer), and various concentrations of hsp90 inhibitor and were incubated at 30°C for 1.5 h. Processing and assembly of P1-2A into pentamers was achieved by the addition of 1 μM 3Cpro to the reaction and incubation at 37°C for 1 h. Following incubation, free radiolabel was removed from reactions by dialysis at 4°C against phosphate-buffered saline (PBS), using mini-dialysis units (Slide-A-Lyzer; Pierce) that had been preblocked with 1% bovine serum albumin in PBS.

Article Title: Proteolytic degradation and potential role of onconeural protein cdr2 in neurodegeneration
Article Snippet: .. In vitro and cell-based calpain cleavage assay For in vitro calpain cleavage assays, the vector encoding T7-tagged mouse cdr2 in pcDNA3 was transcribed and translated in the presence of [35 S]-methionine (Perkin Elmer, Boston, MA, USA) using a TnT Quick coupled transcription/translation system (Promega, Madison, WI, USA) according to the manufacturer's recommendations. .. For cell-based cleavage assay, MN9D cells were lysed in buffer containing 50 mM Tris-HCl, pH 8.0, 2 mM EDTA, and 1% Triton X-100 buffer without protease inhibitor cocktail.

Article Title: Cap-Independent Translation Promotes C. elegans Germ Cell Apoptosis through Apaf-1/CED-4 in a Caspase-Dependent Mechanism
Article Snippet: Protein substrates were labeled with [35 S]-methionine (Perkin Elmer Life Sciences) using TnT T3 coupled reticulocyte lysate system (Promega). .. One microliter of in vitro synthesized product was incubated with 10 µl of recombinant CED-3 at 37°C for 2 h. Control reactions substituted with 10 µl of non-catalytic BSA were prepared in CED-3 reaction buffer.

Article Title: Identification of Unique Antigenic Determinants in the Amino Terminus of IA-2 (ICA512) in Childhood and Adult Autoimmune Diabetes: New Biomarker Development
Article Snippet: .. IA-2ec was in vitro transcribed/translated in the presence of [35 S]methionine (PerkinElmer) using the TNT-coupled rabbit reticulocyte system (Promega, Madison, WI) with T7 RNA polymerase. .. IA-2ec autoantibodies were detected by radiobinding assay using 50% protein A–Sepharose to separate free [35 S]methionine from antibody-bound labeled products.

Article Title: Negative Regulation of Prolactin Receptor Stability and Signaling Mediated by SCFβ-TrCP E3 Ubiquitin Ligase
Article Snippet: For the in vitro ubiquitination assay, GST-PRLR proteins were expressed in bacteria, purified by using glutathione beads, and preincubated (1 μg) with 25-μg extracts from 293T cells treated with PRL (200 ng/ml for 20 min) in a kinase buffer containing 50 mM Tris HCl (pH 7.5), 5 mM MgCl2 , 0.5 mM dithiothreitol, 5 mM NaF, 10 nM okadaic acid, 50 μM ATP, and 10 μM [32 P-γ]ATP for 30 min at 30°C. .. After starvation in DMEM lacking methionine and cysteine and metabolic labeling with a [35 S]methionine-[35 S]cysteine mixture (Perkin-Elmer), cells were harvested at each chase time point with complete DMEM supplemented with FBS (0.5%), PRL (20 ng/ml), and unlabeled methionine and cysteine (2 mM).

Article Title: Opaque-2 Regulates a Complex Gene Network Associated with Cell Differentiation and Storage Functions of Maize Endosperm [OPEN]
Article Snippet: .. Using the resulting constructs as templates, tagged proteins were synthesized in vitro with [35 S]methionine (Perkin-Elmer) using the TnT Quick Coupled Transcription/Translation Systems (Promega) following the manufacturer’s instructions. .. The in vitro pull-down assays were performed as previously described ( ) with minor modifications.

Incubation:

Article Title: Oxidized DJ-1 Interacts with the Mitochondrial Protein BCL-XL
Article Snippet: H1299 cells stably expressing EGFP-Bcl-XL were starved in methionine-free medium and incubated for 30 min. .. The cells were then labeled with 200 μCi/ml [35 S]methionine (PerkinElmer) for 2 h. Pulse-labeling was terminated by incubating cells in fresh medium containing an excess of unlabeled methionine (300 mg/liter), and the cells were harvested at 0, 7, and 14 h. Cells lysates were immunoprecipitated with anti-GFP antibodies (Santa Cruz Biotechnology) and analyzed by SDS-PAGE and autoradiography.

Article Title: MEHMO syndrome mutation EIF2S3-I259M impairs initiator Met-tRNAiMet binding to eukaryotic translation initiation factor eIF2
Article Snippet: .. Aminoacylation reactions with 5 μM synthesized tRNAi Met , 2 mM adenosine triphosphate-Mg2+ , 0.3 μM [35 S]methionine (Perkin Elmer), 10 mM MgCl2 and 1 μM MetRS were incubated in 1× Reaction Buffer (100 mM HEPES-KOH (pH 7.5), 1 mM DTT and 10 mM KCl) for 30 min at 30°C, and aminoacylated Met-tRNAi Met was purified by phenol/chloroform extraction ( ). .. For measurements of tRNA binding, seven 2-fold serial dilutions of eIF2 with final concentrations from 3.125 to 200 nM were incubated for 15 min at 26°C in Binding Assay Buffer (25 mM HEPES-KOH (pH 7.6), 2.5 mM Mg(OAc)2 , 80 mM KOAc (pH 7.6), 2 mM DTT and 0.5 mM guanosine 5′-[β,γ-imido]triphosphate (GDPNP)-Mg2+ ).

Article Title: Functional Importance of Dicer Protein in the Adaptive Cellular Response to Hypoxia *
Article Snippet: .. As a complementary approach, [35 S]methionine (PerkinElmer Life Sciences) was added to the growth medium of confluent HUVEC grown on 12-well plates at a final concentration of 10 mCi/well for the last 0, 20, 40, or 60 min of normoxia or hypoxia after which cells were washed with ice-cold PBS, incubated in 10% TCA for 20 min, washed three times with 100% ethanol, and dried at 45 °C. ..

Article Title: METTL3 promotes translation in human cancer cells
Article Snippet: .. Cells were then incubated for 20 min after supplementation with [35 S]-methionine ([35 S]-Met; 100 mCi/mL; PerkinElmer), washed twice with ice-cold PBS, harvested, and lysed. .. For the quantitation of [35 S]-Met-labeled proteins, cell lysates were subjected to 4–12% Tris-Glycine SDS-PAGE and analyzed by autoradiography.

Article Title: The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers
Article Snippet: .. Reaction mixtures contained 20 ng/μl plasmid DNA, 8 Bq/μl [35 S]methionine (EasyTag; PerkinElmer), and various concentrations of hsp90 inhibitor and were incubated at 30°C for 1.5 h. Processing and assembly of P1-2A into pentamers was achieved by the addition of 1 μM 3Cpro to the reaction and incubation at 37°C for 1 h. Following incubation, free radiolabel was removed from reactions by dialysis at 4°C against phosphate-buffered saline (PBS), using mini-dialysis units (Slide-A-Lyzer; Pierce) that had been preblocked with 1% bovine serum albumin in PBS. ..

Article Title: Proteolytic degradation and potential role of onconeural protein cdr2 in neurodegeneration
Article Snippet: In vitro and cell-based calpain cleavage assay For in vitro calpain cleavage assays, the vector encoding T7-tagged mouse cdr2 in pcDNA3 was transcribed and translated in the presence of [35 S]-methionine (Perkin Elmer, Boston, MA, USA) using a TnT Quick coupled transcription/translation system (Promega, Madison, WI, USA) according to the manufacturer's recommendations. .. [35 S]-cdr2 or cell lysates (50 μ g) were incubated for 1 h at 30 °C in a calpain activation buffer containing 1 mM CaCl2 in the presence or absence of purified m-calpain (0.343 units) or μ -calpain (0.134 units; both from Calbiochem) as recommended by the manufacturer.

Article Title: Cap-Independent Translation Promotes C. elegans Germ Cell Apoptosis through Apaf-1/CED-4 in a Caspase-Dependent Mechanism
Article Snippet: Protein substrates were labeled with [35 S]-methionine (Perkin Elmer Life Sciences) using TnT T3 coupled reticulocyte lysate system (Promega). .. One microliter of in vitro synthesized product was incubated with 10 µl of recombinant CED-3 at 37°C for 2 h. Control reactions substituted with 10 µl of non-catalytic BSA were prepared in CED-3 reaction buffer.

Article Title: Nuclear Aggresomes Form by Fusion of PML-associated Aggregates V⃞
Article Snippet: .. COS-7 cells were transfected with either GFP170* or GFP-250 construct in a six-well plate for 24 h. Cells were then washed in PBS and incubated in methionine-free DMEM for 1 h. Cells were labeled with 200 μCi/ml [35 S]methionine (PerkinElmer Life and Analytical Sciences, Boston, MA) for 60 min. Incorporation was terminated by washing the cells with PBS and chasing with DMEM medium supplemented with 0.2 mM methionine for indicated times. .. At each time point, cells were lysed with RIPA buffer supplemented with protease inhibitor cocktail and 1.0 mM PMSF.

Article Title: Opaque-2 Regulates a Complex Gene Network Associated with Cell Differentiation and Storage Functions of Maize Endosperm [OPEN]
Article Snippet: Using the resulting constructs as templates, tagged proteins were synthesized in vitro with [35 S]methionine (Perkin-Elmer) using the TnT Quick Coupled Transcription/Translation Systems (Promega) following the manufacturer’s instructions. .. Briefly, for a given pair of assayed proteins, the TnT-produced proteins (10 μL each) were mixed, incubated at 30°C for 20 min, and then at 4°C for 20 min while turning on a tube rotator.

Concentration Assay:

Article Title: Functional Importance of Dicer Protein in the Adaptive Cellular Response to Hypoxia *
Article Snippet: .. As a complementary approach, [35 S]methionine (PerkinElmer Life Sciences) was added to the growth medium of confluent HUVEC grown on 12-well plates at a final concentration of 10 mCi/well for the last 0, 20, 40, or 60 min of normoxia or hypoxia after which cells were washed with ice-cold PBS, incubated in 10% TCA for 20 min, washed three times with 100% ethanol, and dried at 45 °C. ..

Lysis:

Article Title: Granzyme B Inhibits Vaccinia Virus Production through Proteolytic Cleavage of Eukaryotic Initiation Factor 4 Gamma 3
Article Snippet: Evaluation of protein synthesis Jurkat cells were pulse-labeled with 33 µCi [35 S]methionine (PerkinElmer) for 1 hour at a density of 3×105 cells/ml. .. To each protein pellet, we added 400 µl of total lysis buffer (2% SDS) and vortexed for 1 min. 5 ml of scintillation fluid was added and samples were counted for 1 min each.

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  • 99
    PerkinElmer s adenosyl l methyl 3 h methionine
    SMYD3 methylates AKT1 in vitro A. Recombinant AKT1 protein was incubated with different concentration of SMYD3 in the presence of <t>S-adenosyl-L-[methyl-</t> 3 H]-methionine, and methylation signal was detected by autoradiography (upper panel). Amounts of loading proteins were evaluated by staining with MemCode™ Reversible Protein Stain (lower panel). The concentration of SMYD3 is 11.5 μM. B. The LC-MS/MS spectrum corresponding to the monomethylated AKT1 9-15 peptide. AKT1 recombinant protein was incubated with SMYD3 and S-adenosyl-L-methionine, followed by separation by SDS-PAGE. An excised AKT1 band from the gel was digested with trypsin and subjected to LC-MS/MS analysis. The methylation site was determined by MASCOT search. The 14 Da increase of the Lys 14 residue was observed. C. The theoretical values of MS/MS fragments ions of the Lys 14 monomethylated AKT1 9-15 peptides are summarized in the table. The abbreviations of fragment ion types were indicated by the MASCOT program ( http://www.matrixscience.com/help/fragmentation_help.html ). The observed ions in Figure 1B were indicated in red letters. D. The biotin-conjugated AKT1 10-44 peptide was incubated with SMYD3 in the presence of S-adenosyl-L-[methyl- 3 H]-methionine, and methylation signal was detected by autoradiography. Amounts of loading proteins were evaluated by staining with MemCode™ Reversible Protein Stain. E. Amino acid sequence alignment of AKT1. Lys 14 is highlighted in red and conserved among various species. Glu 17, which forms an ionic interaction with Lys 14, is shown covering a red hatched rectangle.
    S Adenosyl L Methyl 3 H Methionine, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/s adenosyl l methyl 3 h methionine/product/PerkinElmer
    Average 99 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
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    92
    PerkinElmer s methionine cysteine protein labeling mix
    Constitutive cleavage of PINK1 is mediated by PARL. (a) HeLa cells were transfected with scrambled control siRNA or PARL siRNA. After 4 h incubation with or without 10 µM CCCP, mitochondria were isolated, and mitochondrial <t>protein</t> extracts were assayed for endogenous levels of PINK1 and PARL by immunoblotting. VDAC1 is a mitochondrial marker. (b) MEFs from PARL WT and KO mice were transfected with PINK1-V5/His for 2 d and treated with DMSO or 10 µM CCCP for 4 h. Exogenous PINK1 levels were assayed by immunoblotting. Tubulin is a loading control. (c) PARL WT and KO MEFs were transfected with PINK1-V5/His as in b and treated with DMSO or 10 µM MG132. After 4 h of treatment, cells were fractionated, and exogenous PINK1 levels in mitochondrial fraction were measured with immunoblotting. VDAC1, mitochondrial loading control. (d) [ 35 <t>S]methionine-labeled</t> PINK1 was incubated for different times with mitochondria isolated from PARL WT or KO MEFs in the presence or absence of 1 µM CCCP. After import, samples were treated with or without 5 µg/ml PK. Radiolabeled PINK1 was detected using digital autoradiography. Asterisks, nonspecific bands. (e) [ 35 S]-PINK1 was imported into PARL KO mitochondria for 60 min as in d, and these mitochondria were incubated in the presence or absence of high PK (100 µg/ml) for 10 min. Hsp70, Htra2/Omi, and Tom20 were identified by immunoblotting as markers for mitochondrial matrix, inter membrane space, and outer membrane, respectively. (f) HeLa cells stably expressing YFP-Parkin were transfected with control (siCtrl) or PARL siRNA (siPARL) for 192 h. After transfection, cells were treated with either DMSO or 10 µM CCCP for 1 h, stained with TMRE, and analyzed by live cell imaging. Bars, 20 µm. (g) PARL WT and KO MEFs transfected with PINK1-YFP and mCherry-Parkin were treated with 10 µM DMSO or CCCP for 3 h. Cells (≥50/treatment) were counted for mitochondrial translocation of Parkin. Counting results were represented as mean ± SEM from four replicates. Blue arrowheads, FL PINK1; orange arrowheads, ΔMTS-PINK1; red arrowheads, 52-kD PINK1.
    S Methionine Cysteine Protein Labeling Mix, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    PerkinElmer s radiolabeled methionine
    The degradation of ELK-1 is proteasome-dependent and mediated by FBXO25. The methionine chase labeling experiments were performed using HEK293T cells transfected with the indicated plasmids. After the indicated times, the supernatants were obtained and submitted to immunoprecipitation ( IP ) with anti-ELK-1 antibody. The immunoprecipitated proteins were visualized by Coomassie Blue, and dried gels were film-exposed to protein visualization. A , FBXO25 and not pcDNA3 mediated ELK-1 degradation. B , 35 S-labeled ELK-1-FLAG-His 6 is degraded in the presence of FBXO25 after 12 h of transfection but not by pcDNA3 as indicated in densitometry graphics. C , 35 S-labeled ELK-1 degradation mediated by FBXO25 is proteasome-dependent. D , the proteasome inhibitor epoxomicin protected 35 S-labeled ELK-1-FLAG-His 6 of degradation by FBXO25 as indicated in densitometry graphics. For all experiments n = 2. H.C ., heavy chain. *, the apparent molecular mass of the <t>radiolabeled</t> band ELK-1-FLAG-His 6 on the gels was ascertained by comparison with the mobility of FLAG affinity-purified ELK-1-FLAG-His 6 (data not shown).
    S Radiolabeled Methionine, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 80/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    SMYD3 methylates AKT1 in vitro A. Recombinant AKT1 protein was incubated with different concentration of SMYD3 in the presence of S-adenosyl-L-[methyl- 3 H]-methionine, and methylation signal was detected by autoradiography (upper panel). Amounts of loading proteins were evaluated by staining with MemCode™ Reversible Protein Stain (lower panel). The concentration of SMYD3 is 11.5 μM. B. The LC-MS/MS spectrum corresponding to the monomethylated AKT1 9-15 peptide. AKT1 recombinant protein was incubated with SMYD3 and S-adenosyl-L-methionine, followed by separation by SDS-PAGE. An excised AKT1 band from the gel was digested with trypsin and subjected to LC-MS/MS analysis. The methylation site was determined by MASCOT search. The 14 Da increase of the Lys 14 residue was observed. C. The theoretical values of MS/MS fragments ions of the Lys 14 monomethylated AKT1 9-15 peptides are summarized in the table. The abbreviations of fragment ion types were indicated by the MASCOT program ( http://www.matrixscience.com/help/fragmentation_help.html ). The observed ions in Figure 1B were indicated in red letters. D. The biotin-conjugated AKT1 10-44 peptide was incubated with SMYD3 in the presence of S-adenosyl-L-[methyl- 3 H]-methionine, and methylation signal was detected by autoradiography. Amounts of loading proteins were evaluated by staining with MemCode™ Reversible Protein Stain. E. Amino acid sequence alignment of AKT1. Lys 14 is highlighted in red and conserved among various species. Glu 17, which forms an ionic interaction with Lys 14, is shown covering a red hatched rectangle.

    Journal: Oncotarget

    Article Title: SMYD3-mediated lysine methylation in the PH domain is critical for activation of AKT1

    doi: 10.18632/oncotarget.11898

    Figure Lengend Snippet: SMYD3 methylates AKT1 in vitro A. Recombinant AKT1 protein was incubated with different concentration of SMYD3 in the presence of S-adenosyl-L-[methyl- 3 H]-methionine, and methylation signal was detected by autoradiography (upper panel). Amounts of loading proteins were evaluated by staining with MemCode™ Reversible Protein Stain (lower panel). The concentration of SMYD3 is 11.5 μM. B. The LC-MS/MS spectrum corresponding to the monomethylated AKT1 9-15 peptide. AKT1 recombinant protein was incubated with SMYD3 and S-adenosyl-L-methionine, followed by separation by SDS-PAGE. An excised AKT1 band from the gel was digested with trypsin and subjected to LC-MS/MS analysis. The methylation site was determined by MASCOT search. The 14 Da increase of the Lys 14 residue was observed. C. The theoretical values of MS/MS fragments ions of the Lys 14 monomethylated AKT1 9-15 peptides are summarized in the table. The abbreviations of fragment ion types were indicated by the MASCOT program ( http://www.matrixscience.com/help/fragmentation_help.html ). The observed ions in Figure 1B were indicated in red letters. D. The biotin-conjugated AKT1 10-44 peptide was incubated with SMYD3 in the presence of S-adenosyl-L-[methyl- 3 H]-methionine, and methylation signal was detected by autoradiography. Amounts of loading proteins were evaluated by staining with MemCode™ Reversible Protein Stain. E. Amino acid sequence alignment of AKT1. Lys 14 is highlighted in red and conserved among various species. Glu 17, which forms an ionic interaction with Lys 14, is shown covering a red hatched rectangle.

    Article Snippet: In vitro methyltransferase assay For the in vitro methyltransferase assay, recombinant AKT1 (P2999, Thermo Fisher Scientific) was incubated with recombinant SMYD3 enzyme using 2 μCi S-adenosyl-L-[methyl-3 H]-methionine (SAM; PerkinElmer) as the methyl donor in a mixture of 10 μl of methylase activity buffer (50 mM Tris-HCl at pH8.8, 10 mM dithiothreitol (DTT) and 10 mM MgCl2 ), for 2 hours at 30°C [ – ].

    Techniques: In Vitro, Recombinant, Incubation, Concentration Assay, Methylation, Autoradiography, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, SDS Page, Sequencing

    Automethylation of SUV39H2 and methylation of substrate proteins are exclusively correlated A-B. Recombinant SUV39H2 proteins were individually incubated with increasing amount of histone H3 protein (A) or LSD1 protein (B) in the presence of S-adenosyl-L-[methyl- 3 H]-methionine. The reaction mixtures were separated by SDS-PAGE, and the intensities of methylated proteins were detected by autoradiography, whereas the loading amounts of proteins were stained with Coomassie Brilliant Blue. C. The automethylation site (lysine 392) is located in a flexible loop (black dot line), which is suggested to form a part of the substrate lysine pocket. Pre-SET, SET and Post-SET domains are colored blue, orange, and green, respectively. SAM is shown as yellow stick and four cysteine residues corresponding to zinc binding are highlighted with red color.

    Journal: Oncotarget

    Article Title: Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins

    doi: 10.18632/oncotarget.8072

    Figure Lengend Snippet: Automethylation of SUV39H2 and methylation of substrate proteins are exclusively correlated A-B. Recombinant SUV39H2 proteins were individually incubated with increasing amount of histone H3 protein (A) or LSD1 protein (B) in the presence of S-adenosyl-L-[methyl- 3 H]-methionine. The reaction mixtures were separated by SDS-PAGE, and the intensities of methylated proteins were detected by autoradiography, whereas the loading amounts of proteins were stained with Coomassie Brilliant Blue. C. The automethylation site (lysine 392) is located in a flexible loop (black dot line), which is suggested to form a part of the substrate lysine pocket. Pre-SET, SET and Post-SET domains are colored blue, orange, and green, respectively. SAM is shown as yellow stick and four cysteine residues corresponding to zinc binding are highlighted with red color.

    Article Snippet: Briefly, purified His-SUV39H2 expressed in Baculovirus infected insect cells or purified His-SUV39H2 with purified histone H3 expressed in E.coli or purified LSD1 expressed in Baculovirus infected insect cells was incubated in 50 mM Tris-HCl (pH 8.8) buffer with 1.0 μCi/ml S-adenosyl-L-[methyl-3 H]-methionine (Perkin Elmer, Waltham, MA) for 2 hours at 30°C.

    Techniques: Methylation, Recombinant, Incubation, SDS Page, Autoradiography, Staining, Binding Assay

    Automethylation of SUV39H2 negatively regulates SUV39H2 methylation activities to other substrates A-B. Recombinant H3 protein (A) or recombinant LSD1 protein (B) was reacted with equal amount of recombinant SUV39H2 proteins or hyper-automethylated SUV39H2 (pre-incubated with S-adenosyl-L-[methyl- 3 H]-methionine for 4 hours). The intensity of methylated LSD1 protein for each sample was quantified by GS-800™ calibrated densitometer (Bio-Rad) and plotted (right panel). All error bars indicate SEM of two independent experiments. Values are relative to the intensity obtained from the reaction of 10 pmol LSD1 and hypomethylated SUV39H2. The loading amounts of proteins were stained with Coomassie Brilliant Blue (A) and MemCode™ Reversible Protein Stain (B). C. 293T cells co-expressing HA-LSD1 with FLAG-SUV39H2-WT, FLAG-K392A or FLAG-K392R were labelled with L-[methyl- 3 H]-methionine in vivo . Cell lysates were immunoprecipitated with anti-HA agarose (Sigma-Aldrich) and methylated LSD1 was visualized by autoradiography. Immunoprecipitates were immunoblotted with anti-HA antibody as an internal control.

    Journal: Oncotarget

    Article Title: Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins

    doi: 10.18632/oncotarget.8072

    Figure Lengend Snippet: Automethylation of SUV39H2 negatively regulates SUV39H2 methylation activities to other substrates A-B. Recombinant H3 protein (A) or recombinant LSD1 protein (B) was reacted with equal amount of recombinant SUV39H2 proteins or hyper-automethylated SUV39H2 (pre-incubated with S-adenosyl-L-[methyl- 3 H]-methionine for 4 hours). The intensity of methylated LSD1 protein for each sample was quantified by GS-800™ calibrated densitometer (Bio-Rad) and plotted (right panel). All error bars indicate SEM of two independent experiments. Values are relative to the intensity obtained from the reaction of 10 pmol LSD1 and hypomethylated SUV39H2. The loading amounts of proteins were stained with Coomassie Brilliant Blue (A) and MemCode™ Reversible Protein Stain (B). C. 293T cells co-expressing HA-LSD1 with FLAG-SUV39H2-WT, FLAG-K392A or FLAG-K392R were labelled with L-[methyl- 3 H]-methionine in vivo . Cell lysates were immunoprecipitated with anti-HA agarose (Sigma-Aldrich) and methylated LSD1 was visualized by autoradiography. Immunoprecipitates were immunoblotted with anti-HA antibody as an internal control.

    Article Snippet: Briefly, purified His-SUV39H2 expressed in Baculovirus infected insect cells or purified His-SUV39H2 with purified histone H3 expressed in E.coli or purified LSD1 expressed in Baculovirus infected insect cells was incubated in 50 mM Tris-HCl (pH 8.8) buffer with 1.0 μCi/ml S-adenosyl-L-[methyl-3 H]-methionine (Perkin Elmer, Waltham, MA) for 2 hours at 30°C.

    Techniques: Methylation, Recombinant, Incubation, Staining, Expressing, In Vivo, Immunoprecipitation, Autoradiography

    Constitutive cleavage of PINK1 is mediated by PARL. (a) HeLa cells were transfected with scrambled control siRNA or PARL siRNA. After 4 h incubation with or without 10 µM CCCP, mitochondria were isolated, and mitochondrial protein extracts were assayed for endogenous levels of PINK1 and PARL by immunoblotting. VDAC1 is a mitochondrial marker. (b) MEFs from PARL WT and KO mice were transfected with PINK1-V5/His for 2 d and treated with DMSO or 10 µM CCCP for 4 h. Exogenous PINK1 levels were assayed by immunoblotting. Tubulin is a loading control. (c) PARL WT and KO MEFs were transfected with PINK1-V5/His as in b and treated with DMSO or 10 µM MG132. After 4 h of treatment, cells were fractionated, and exogenous PINK1 levels in mitochondrial fraction were measured with immunoblotting. VDAC1, mitochondrial loading control. (d) [ 35 S]methionine-labeled PINK1 was incubated for different times with mitochondria isolated from PARL WT or KO MEFs in the presence or absence of 1 µM CCCP. After import, samples were treated with or without 5 µg/ml PK. Radiolabeled PINK1 was detected using digital autoradiography. Asterisks, nonspecific bands. (e) [ 35 S]-PINK1 was imported into PARL KO mitochondria for 60 min as in d, and these mitochondria were incubated in the presence or absence of high PK (100 µg/ml) for 10 min. Hsp70, Htra2/Omi, and Tom20 were identified by immunoblotting as markers for mitochondrial matrix, inter membrane space, and outer membrane, respectively. (f) HeLa cells stably expressing YFP-Parkin were transfected with control (siCtrl) or PARL siRNA (siPARL) for 192 h. After transfection, cells were treated with either DMSO or 10 µM CCCP for 1 h, stained with TMRE, and analyzed by live cell imaging. Bars, 20 µm. (g) PARL WT and KO MEFs transfected with PINK1-YFP and mCherry-Parkin were treated with 10 µM DMSO or CCCP for 3 h. Cells (≥50/treatment) were counted for mitochondrial translocation of Parkin. Counting results were represented as mean ± SEM from four replicates. Blue arrowheads, FL PINK1; orange arrowheads, ΔMTS-PINK1; red arrowheads, 52-kD PINK1.

    Journal: The Journal of Cell Biology

    Article Title: Mitochondrial membrane potential regulates PINK1 import and proteolytic destabilization by PARL

    doi: 10.1083/jcb.201008084

    Figure Lengend Snippet: Constitutive cleavage of PINK1 is mediated by PARL. (a) HeLa cells were transfected with scrambled control siRNA or PARL siRNA. After 4 h incubation with or without 10 µM CCCP, mitochondria were isolated, and mitochondrial protein extracts were assayed for endogenous levels of PINK1 and PARL by immunoblotting. VDAC1 is a mitochondrial marker. (b) MEFs from PARL WT and KO mice were transfected with PINK1-V5/His for 2 d and treated with DMSO or 10 µM CCCP for 4 h. Exogenous PINK1 levels were assayed by immunoblotting. Tubulin is a loading control. (c) PARL WT and KO MEFs were transfected with PINK1-V5/His as in b and treated with DMSO or 10 µM MG132. After 4 h of treatment, cells were fractionated, and exogenous PINK1 levels in mitochondrial fraction were measured with immunoblotting. VDAC1, mitochondrial loading control. (d) [ 35 S]methionine-labeled PINK1 was incubated for different times with mitochondria isolated from PARL WT or KO MEFs in the presence or absence of 1 µM CCCP. After import, samples were treated with or without 5 µg/ml PK. Radiolabeled PINK1 was detected using digital autoradiography. Asterisks, nonspecific bands. (e) [ 35 S]-PINK1 was imported into PARL KO mitochondria for 60 min as in d, and these mitochondria were incubated in the presence or absence of high PK (100 µg/ml) for 10 min. Hsp70, Htra2/Omi, and Tom20 were identified by immunoblotting as markers for mitochondrial matrix, inter membrane space, and outer membrane, respectively. (f) HeLa cells stably expressing YFP-Parkin were transfected with control (siCtrl) or PARL siRNA (siPARL) for 192 h. After transfection, cells were treated with either DMSO or 10 µM CCCP for 1 h, stained with TMRE, and analyzed by live cell imaging. Bars, 20 µm. (g) PARL WT and KO MEFs transfected with PINK1-YFP and mCherry-Parkin were treated with 10 µM DMSO or CCCP for 3 h. Cells (≥50/treatment) were counted for mitochondrial translocation of Parkin. Counting results were represented as mean ± SEM from four replicates. Blue arrowheads, FL PINK1; orange arrowheads, ΔMTS-PINK1; red arrowheads, 52-kD PINK1.

    Article Snippet: In vitro import of PINK1 into isolated mitochondria Generation of radiolabeled PINK1 precursor was performed by in vitro transcription followed by translation using rabbit reticulocyte lysates (Promega) in the presence of [35 S]methionine/cysteine protein-labeling mix (PerkinElmer) as previously described ( ).

    Techniques: Transfection, Incubation, Isolation, Marker, Mouse Assay, Labeling, Autoradiography, Stable Transfection, Expressing, Staining, Live Cell Imaging, Translocation Assay

    The degradation of ELK-1 is proteasome-dependent and mediated by FBXO25. The methionine chase labeling experiments were performed using HEK293T cells transfected with the indicated plasmids. After the indicated times, the supernatants were obtained and submitted to immunoprecipitation ( IP ) with anti-ELK-1 antibody. The immunoprecipitated proteins were visualized by Coomassie Blue, and dried gels were film-exposed to protein visualization. A , FBXO25 and not pcDNA3 mediated ELK-1 degradation. B , 35 S-labeled ELK-1-FLAG-His 6 is degraded in the presence of FBXO25 after 12 h of transfection but not by pcDNA3 as indicated in densitometry graphics. C , 35 S-labeled ELK-1 degradation mediated by FBXO25 is proteasome-dependent. D , the proteasome inhibitor epoxomicin protected 35 S-labeled ELK-1-FLAG-His 6 of degradation by FBXO25 as indicated in densitometry graphics. For all experiments n = 2. H.C ., heavy chain. *, the apparent molecular mass of the radiolabeled band ELK-1-FLAG-His 6 on the gels was ascertained by comparison with the mobility of FLAG affinity-purified ELK-1-FLAG-His 6 (data not shown).

    Journal: The Journal of Biological Chemistry

    Article Title: The F-box Protein FBXO25 Promotes the Proteasome-dependent Degradation of ELK-1 Protein *

    doi: 10.1074/jbc.M113.504308

    Figure Lengend Snippet: The degradation of ELK-1 is proteasome-dependent and mediated by FBXO25. The methionine chase labeling experiments were performed using HEK293T cells transfected with the indicated plasmids. After the indicated times, the supernatants were obtained and submitted to immunoprecipitation ( IP ) with anti-ELK-1 antibody. The immunoprecipitated proteins were visualized by Coomassie Blue, and dried gels were film-exposed to protein visualization. A , FBXO25 and not pcDNA3 mediated ELK-1 degradation. B , 35 S-labeled ELK-1-FLAG-His 6 is degraded in the presence of FBXO25 after 12 h of transfection but not by pcDNA3 as indicated in densitometry graphics. C , 35 S-labeled ELK-1 degradation mediated by FBXO25 is proteasome-dependent. D , the proteasome inhibitor epoxomicin protected 35 S-labeled ELK-1-FLAG-His 6 of degradation by FBXO25 as indicated in densitometry graphics. For all experiments n = 2. H.C ., heavy chain. *, the apparent molecular mass of the radiolabeled band ELK-1-FLAG-His 6 on the gels was ascertained by comparison with the mobility of FLAG affinity-purified ELK-1-FLAG-His 6 (data not shown).

    Article Snippet: After 24 h, the cells were washed with PBS and DMEM without methionine (Sigma-Aldrich) and kept in this medium for 2 h. Then the cells were pulsed for 1 h with 150 μCi/well 35 S-radiolabeled methionine (Express labeling mix 35 S; PerkinElmer Life Sciences).

    Techniques: Labeling, Transfection, Immunoprecipitation, Affinity Purification