Recombinant myc-TLK1 wild-type (wt) or kinase-inactive (ki) were purified from Sf9 insect cells, infected with recombinant baculovirus34 (link) using protein G sepharose beads (GE Healthcare) coupled to myc antibodies. Myc-TLK1 was eluted in low pH buffer (100 mM glycin pH 2.5, 1 mM DTT, 2.5 mM NaF), and pH was adjusted by addition of 1M Tris pH 8.0 (supplemented with 1 mM DTT, 2.5 mM NaF). In vitro TLK1 kinase assays were performed essentially as described34 (link),35 (link). In brief, myc-TLK1 was incubated in 25 μl kinase assay buffer (KAB - 50 mM HEPES pH 7.2, 10 mM MgCl2, 5 mM MnCl2, 2.5 mM EGTA, 1 mM DTT, 1 mM NaF, 10 μM ATP, 10μCi/μl [γ32P]-ATP) with 1 μg GST-Asf1a for 30 min and 30°C. Kinase reactions were stopped by the addition of 4xLSB, and reaction products were resolved on a 12% SDS-PAGE. [32P] incorporation was quantified on a PhosphorImager FLA3000 (Fujifilm, Tokyo). For identification of TLK1 phospho-sites in Asf1 (a and b) by mass spectrometry, the kinase reaction contained 50 μM ATP, 6μg of recombinant Asf1 (a or b) and myc-TLK1 (2μg). Reaction products were resolved on a 15 % SDS-PAGE gel and stained with Coomassie Blue. Slow-migrating bands were cut and analyzed by LC-MS/MS.
Fla3000 phosphorimager
Manufactured by Fujifilm
Sourced in Japan
The FLA3000 phosphorimager is a lab equipment product from Fujifilm. It is designed for the detection and quantification of radioactive signals in gels, blots, and other sample types. The FLA3000 utilizes a laser-based scanning technology to capture high-resolution images of radioactive samples.
Automatically generated - may contain errors
Lab products found in correlation
Image gauge software, by Fujifilm (4 mentions)
Protein g sepharose bead, by GE Healthcare (2 mentions)
Genescreen plus, by PerkinElmer (2 mentions)
Anti v5 antibody, by Thermo Fisher Scientific (2 mentions)
35s cys 35s met, by PerkinElmer (2 mentions)
Protein a or protein g coated beads, by Thermo Fisher Scientific (2 mentions)
Bas mpphosphor, by Fujifilm (2 mentions)
Pngase f, by New England Biolabs (2 mentions)
Histone h1, by Roche (2 mentions)
γ 32p atp, by PerkinElmer (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Baker s yeast, by Merck Group (1 mentions)
Iblot, by Thermo Fisher Scientific (1 mentions)
Aida image analyzer, by Fujifilm (1 mentions)
Megaprime dna labeling system, by GE Healthcare (1 mentions)
Zeta probe gt genomic tested blotting membranes, by Bio-Rad (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Trapeze telomerase detection kit, by Merck Group (1 mentions)
Sybr gold nucleic acid gel stain, by Thermo Fisher Scientific (1 mentions)
Bas ms imaging plate, by Fujifilm (1 mentions)
37 protocols using fla3000 phosphorimager
1
Purification and Kinase Assay of TLK1
2
Mycelial Protein Extraction and Analysis
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Spores were inoculated at a density of 1×106 to 2×106 spores/ml, in 100 ml of liquid ACM, and grown for 16 hr at 37°C. Next, media was buffered to pH 5.0 with 100 mM glycolic acid pH 5.0 or to pH 8.0 with 100 mM Tris-HCl pH 8.0, pH shifts were performed for 1 hour. 10 mg of protein was extracted from washed mycelia as described previously [73] (link). Protein concentrations were determined using the Bradford assay [74] (link). The ipnA2 probe was synthesised and labelled as described previously [27] (link), [73] (link). Densitometry data were obtained by measuring pixel intensity/mm2 for the relevant bands using a Phosphorimager FLA-3000 (FujiFilm) and Multi-Gauge V3.0 software.
3
Northern Blot Analysis of Gene Expression
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Total RNA was purified as described [102 (link)]. For blotting, 5 and 10 μg of total RNA were separated in 1% denaturing agarose gel, transferred to nylon membrane (GeneScreen Plus; Perkin-Elmer), UV cross-linked, baked and hybridized at 42°C overnight with random primed 32 P-DNA fragments of genes of interest. Hybridization solution was 5X Denhardt’s, 5X SSC, 50% formamide, 0.2% SDS, 5 mM EDTA, 100 μg/ml total yeast RNA (Baker’s yeast, Sigma). Quantitation was by PhosphorImager FLA-3000 (Fuji Film). For sequential probings, signal was stripped, monitored for loss of 32 P before next probing.
4
Purification and Kinase Assay of TLK1
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Recombinant myc-TLK1 wild-type (wt) or kinase-inactive (ki) were purified from Sf9 insect cells, infected with recombinant baculovirus34 (link) using protein G sepharose beads (GE Healthcare) coupled to myc antibodies. Myc-TLK1 was eluted in low pH buffer (100 mM glycin pH 2.5, 1 mM DTT, 2.5 mM NaF), and pH was adjusted by addition of 1M Tris pH 8.0 (supplemented with 1 mM DTT, 2.5 mM NaF). In vitro TLK1 kinase assays were performed essentially as described34 (link),35 (link). In brief, myc-TLK1 was incubated in 25 μl kinase assay buffer (KAB - 50 mM HEPES pH 7.2, 10 mM MgCl2, 5 mM MnCl2, 2.5 mM EGTA, 1 mM DTT, 1 mM NaF, 10 μM ATP, 10μCi/μl [γ32P]-ATP) with 1 μg GST-Asf1a for 30 min and 30°C. Kinase reactions were stopped by the addition of 4xLSB, and reaction products were resolved on a 12% SDS-PAGE. [32P] incorporation was quantified on a PhosphorImager FLA3000 (Fujifilm, Tokyo). For identification of TLK1 phospho-sites in Asf1 (a and b) by mass spectrometry, the kinase reaction contained 50 μM ATP, 6μg of recombinant Asf1 (a or b) and myc-TLK1 (2μg). Reaction products were resolved on a 15 % SDS-PAGE gel and stained with Coomassie Blue. Slow-migrating bands were cut and analyzed by LC-MS/MS.
5
Quantification of i6A37 tRNA Modification
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Total RNAs were isolated and separated on 10% polyacrylamide–urea gel, transferred to nylon membrane (GeneScreen Plus; PerkinElmer) using IBlot (Invitrogen). The membrane was then UV cross-linked and vacuum dried at 80°C for 2 h. It was hybridized with specific 32P-labeled DNA oligos as described previously (Lamichhane et al. 2011 (link)). Blots were exposed to a phosphorimager screen, scanned, and quantified using a Phosphorimager FLA-3000 (Fujifilm). The PHA6 assay was used to assess the level of i6A37 modification (Lamichhane et al. 2013a (link),b (link); Yarham et al. 2014 (link)). Quantification of the efficiency of i6A37 modification used the formula: % modification = [1 − (ACLtit1+/TSLtit1+)/(ACLtit1-Δ/TSLtit1-Δ)] × 100, where “ACL” indicates the ACL probe and “TSL” indicates the TSL probe. As previously described, this formula includes an internal normalization from the tit1-Δ samples (Lamichhane et al. 2013a (link),b (link); Yarham et al. 2014 (link)). However, it should be noted that given the principle of PHA6 assay, which depends on the amount of hypomodified adenosine (A37), the modification ratio calculated by this formula thus represents the relative amount of i6A, but not absolute modification rate.
6
Ribosome Structural Changes in Oxidative Stress
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wt and rcm1-knockout (rcm1Δ) yeast cells were grown in SC medium at 30 °C to a final OD600 of 0.6–0.8. Then 0.4 mM H2O2 was added for 30 or 90 min and cells were collected at 6,000g. The 80S ribosomes from unstressed and stressed cells were isolated as described previously48 (link). Before primer extension analysis, 10 pmol of ribosomes were incubated with buffer (20 mM HEPES/KOH pH 7.6, 6 mM MgAc2, 150 mM NH4Cl, 2 mM spermidine, 0.05 mM spermine, 4 mM beta-mercaptoethanol) for 10 min at 37 °C. Chemical probing with DMS at 37 °C for 15 min was performed as described49 (link). To monitor the DMS-reactive sites in domain IV of 25S rRNAs, primer extension analysis (primers used: 5′-GTTCCCTTGGCTGTGGTTTC-3′; 5′-TCTCGTTAATCCATTCATG-3′) was performed as previously described50 (link). Primer extension products were separated on 8% denaturing polyacrylamide gels and visualized by using a PhosphorImager (FLA-3000; Fuji Photo Film) and quantified with the densitometric programme Aida Image Analyzer.
7
Time-resolved Translocation Assay with Disulfide-stabilized Precursors
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A time course of translocation assay was performed before each AFM experiment using the precursors containing disulfide-stabilized loops. Translocation of 1 μM oxidized pOmpA or pGBP, labeled with a 14C-l -amino acid mixture, into proteoliposomes was carried out at 30°C under conditions of limiting SecY (1 μM) with SecA (1.2 μM dimer), SecB (1 μM tetramer), and EGTA (1 mM) to prevent pGBP from folding, as previously described (40 (link)), with the following modifications: An ATP-regenerating system consisting of 7.5 mM phosphocreatine and creatine phosphokinase (37 mg/ml) was present in the reaction. In addition, DTT was omitted from the reaction mix, and different nucleotides were added in the reaction mixture in place of ATP. In the case of ADP or ADPAlF, the ATP-regenerating system was omitted to prevent generation of ATP. A volume of sample selected for translocation quantification was subjected to proteinase K digestion. The radioactivity in the protein bands corresponding to protected precursors in the gels of the translocation assays was measured using a Fujifilm FLA 3000 PhosphorImager in the linear range of its response.
8
Quantifying Radiolabeled SecB Protein
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14C-SecB was precipitated at two different concentrations: 80 μg ml-1 (HIGH) and 0.34 μg ml-1 (LOW) by addition of trichloroacetic acid (TCA) to a final concentration of 11% (wt wt-1). The precipitates were collected by centrifugation (16,000 × g, 15 min at 10°C, Eppendorf centrifuge), washed with acetone and suspended in sample buffer. 14C-SecB that had not been precipitated with TCA was loaded on 14% (wt vol-1) polyacrylamide gels (0.54 mm thickness) for SDS-polyacrylamide gel electrophoresis at different concentrations to prepare a standard. The TCA precipitated samples were loaded to same amount assuming 100% recovery. The quantities of 14C-SecB on the gels were measured using a Fujifilm FLA 3000 phosphorimager in the linear range of its response.
9
Quantitative Analysis of PD-L1 Turnover
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V5-tagged PD-L1 transduced CMTM6 overexpressing A375 cells, and
V5-tagged PD-L1 transduced CMTM6 knockout A375 cells were cultured in
methionine- and cysteine-free medium for 1h at 37°C. Cells were then
pulse labeled with 0.5 mCi/ml [35S]Cys/[35S]Met
(PerkinElmer) for 1 hour. Cells were washed with PBS to remove residual
[35S]Cys/[35S]Met, and then cultured in regular medium
with extra ‘cold’ methionine and cysteine for 0, 1, 2, 3 and 6h.
Cell samples were lysed and used for immunoprecipitation with anti-V5 antibody
(ThermoFisher) immobilized on protein A or protein G coated beads
(ThermoFisher). Immunoprecipitates were either left untreated or treated with
EndoH or PNGaseF (New England Biolabs), according to the manufacturer’s
instructions.
Immunoprecipitates were run on NuPAGE 4-12% gels. Gels were treated with
1M NaSalicylate pH5.6 before drying, and then analysed on Fujifilm BAS-MP
phosphor imager screens. Quantification was performed using a Fujifilm FLA-3000
phosphorimager and AIDA image analyzer software. Gels were exposed to film using
intensifier screens at -80 C.
V5-tagged PD-L1 transduced CMTM6 knockout A375 cells were cultured in
methionine- and cysteine-free medium for 1h at 37°C. Cells were then
pulse labeled with 0.5 mCi/ml [35S]Cys/[35S]Met
(PerkinElmer) for 1 hour. Cells were washed with PBS to remove residual
[35S]Cys/[35S]Met, and then cultured in regular medium
with extra ‘cold’ methionine and cysteine for 0, 1, 2, 3 and 6h.
Cell samples were lysed and used for immunoprecipitation with anti-V5 antibody
(ThermoFisher) immobilized on protein A or protein G coated beads
(ThermoFisher). Immunoprecipitates were either left untreated or treated with
EndoH or PNGaseF (New England Biolabs), according to the manufacturer’s
instructions.
Immunoprecipitates were run on NuPAGE 4-12% gels. Gels were treated with
1M NaSalicylate pH5.6 before drying, and then analysed on Fujifilm BAS-MP
phosphor imager screens. Quantification was performed using a Fujifilm FLA-3000
phosphorimager and AIDA image analyzer software. Gels were exposed to film using
intensifier screens at -80 C.
10
Northern Blot & Real-Time PCR for WIG-1 Expression
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For Northern blot analyses cells were harvested by trypsination and total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer's protocol. RNA was separated in 1% agarose/formamide gels and blotted to Zeta-Probe GT Genomic Tested Blotting membranes (Bio-Rad, Hercules, CA, USA). Probes used for hybridization were the coding part of WIG-1 cDNA and a 500 bp PCR fragment of beta-actin labeled with the Megaprime DNA labeling System (GE Healthcare, Uppsala, Sweden). Hybridization was performed in ULTRA hybridization buffer (Life Technologies, Stockholm, Sweden) and the signals were detected and analyzed in a FLA-3000 phosphorimager (Fujifilm, Stockholm, Sweden). Normal human fibroblasts were used as control with 18S RNA as a reference for the analysis.
WIG-1 expression was also analysed by real-time PCR using HPRT1 as reference gene and SYBR Green based LightCycler PCR as further described in the Supplementary material.
WIG-1 expression was also analysed by real-time PCR using HPRT1 as reference gene and SYBR Green based LightCycler PCR as further described in the Supplementary material.
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