Yes1 recombinant human protein was obtained from Life technologies (A15557). To purify OCT2 proteins, FLAG-tagged wild-type or mutant OCT2 constructs (described in the site-directed mutagenesis section) were subcloned into pT7CFE1-CHis plasmid (Thermo Fischer). These constructs were then used for in vitro translation using a HeLa cell lysate-based Kit (1-Step Human Coupled IVT Kit – DNA; 88881, Life Technologies). The in vitro translated proteins were then purified using His Pur cobalt spin columns (Thermo Scientific). For in vitro kinase assays, recombinant Yes1 and purified OCT2 proteins were incubated in a kinase buffer (Cell Signaling, 9802) supplemented with cold ATP (Cell signaling, 9804) at 30 °C for 30 min. After the incubation period, the reaction was terminated and OCT2 proteins were immunoprecipitated by FLAG tagged beads (described in the Protein Analysis section) followed by western blot analysis to determine OCT2 tyrosine phosphorylation.
Pt7cfe1 chis plasmid
Manufactured by Thermo Fisher Scientific
The PT7CFE1-CHis plasmid is a laboratory tool used for the expression and purification of recombinant proteins. It contains a T7 promoter and a His-tag sequence, which facilitate the production and isolation of the target protein. The core function of this plasmid is to enable the expression and purification of recombinant proteins in a controlled laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Kinase buffer, by Cell Signaling Technology (3 mentions)
1 step human coupled ivt kit dna, by Thermo Fisher Scientific (3 mentions)
His pur cobalt spin columns, by Thermo Fisher Scientific (3 mentions)
Adp glo kinase assay kit, by Promega (2 mentions)
Prism 9, by GraphPad (2 mentions)
In fusion cloning, by Takara Bio (1 mentions)
4 protocols using pt7cfe1 chis plasmid
1
Purification and Phosphorylation of Recombinant OCT2
2
In Vitro CDKL5 Kinase Assay
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Methods used for these assays have been described.28 (link) Briefly, human FLAG-tagged CDKL5 WT or kinase dead (KD, CDKL5 K42R) constructs were subcloned into pT7CFE1-CHis plasmid (Thermo Fisher). These constructs were then used for in vitro translation using a HeLa cell lysate-based Kit (1-Step Human Coupled IVT Kit—DNA, 88881, Life Technologies). The in vitro-translated proteins were then purified using His Pur cobalt spin columns (Thermo Scientific). For in vitro kinase assays, recombinant CDKL5 and myelin basic protein (Active Motif, 31314) as a substrate were incubated in a kinase buffer (Cell Signaling, 9802) supplemented with or without 50 µM adenosine 5′-triphosphate (ATP) at 30 °C for 30 minutes followed by kinase assays using ADP-Glo Kinase Assay kit (Promega). Data were analyzed using GraphPad Prism 9. Each assay was run in triplicate (n = 3) and mean values are graphed in Figure 7 . All error bars in Figure 7 are standard deviation (SD). Statistical analysis was done using one-way ANOVA with Dunnett’s multiple comparisons test. Statistical methods and p-values are mentioned in the Figure 7 legend. Thresholds for significance were placed at ***p <0.0001. Non-significant statistics were not indicated.
3
In Vitro Kinase Assay for CDKL5
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Methods used for these assays
have been described.30 (link) Briefly, human FLAG-tagged
CDKL5 WT or KD (CDKL5 K42R) constructs were subcloned into pT7CFE1-CHis
plasmid (Thermo Fisher). These constructs were then used for in vitro
translation using a HeLa cell lysate-based kit (1-Step Human Coupled
IVT Kit—DNA, 88881, Life Technologies). The in vitro-translated
proteins were then purified using HisPur cobalt spin columns (Thermo
Scientific). For in vitro kinase assays, recombinant CDKL5 and myelin
basic protein (Active Motif, 31314) as a substrate were incubated
in a kinase buffer (Cell Signaling, 9802) supplemented with or without
50 μM adenosine 5′-triphosphate (ATP) at 30 °C for
30 min followed by kinase assays using the ADP-Glo kinase assay
kit (Promega). Data were analyzed using GraphPad Prism 9. Each assay
was run in triplicate (n = 3), and the mean values
are graphed inFigure 7 . All error bars in Figure 7 are standard deviation (SD). Statistical analysis was done
using one-way ANOVA with Dunnett’s multiple comparisons test.
Statistical methods and p-values are mentioned in
theFigure 7 legend.
Thresholds for significance were placed at ***p <
0.0001. Non-significant statistics were not indicated.
have been described.30 (link) Briefly, human FLAG-tagged
CDKL5 WT or KD (CDKL5 K42R) constructs were subcloned into pT7CFE1-CHis
plasmid (Thermo Fisher). These constructs were then used for in vitro
translation using a HeLa cell lysate-based kit (1-Step Human Coupled
IVT Kit—DNA, 88881, Life Technologies). The in vitro-translated
proteins were then purified using HisPur cobalt spin columns (Thermo
Scientific). For in vitro kinase assays, recombinant CDKL5 and myelin
basic protein (Active Motif, 31314) as a substrate were incubated
in a kinase buffer (Cell Signaling, 9802) supplemented with or without
50 μM adenosine 5′-triphosphate (ATP) at 30 °C for
30 min followed by kinase assays using the ADP-Glo kinase assay
kit (Promega). Data were analyzed using GraphPad Prism 9. Each assay
was run in triplicate (n = 3), and the mean values
are graphed in
using one-way ANOVA with Dunnett’s multiple comparisons test.
Statistical methods and p-values are mentioned in
the
Thresholds for significance were placed at ***p <
0.0001. Non-significant statistics were not indicated.
4
Dual-Luciferase Reporter Construction
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The DNA template sequences of the different reporters used in this study are depicted in sup. Fig. 3.The p200-6xMS2 plasmid encoding the RLuc reporter used for in vitro transcription is described in (19) . We created the dual-luciferase reporter pCRIIRLuc-UAA-FLucA80 by ligating NotI-KpnI digested pCRII-hRLuc-200bp-A80 ( 16) and the PCR-amplified Luc ORFs from the plasmid p2luc-UAA (20) (digested with the same restriction enzymes). The ECMV IRES sequence was subcloned into pCRII-RLuc-UAA-FLuc-A80 from the pT7CFE1-CHis plasmid (Thermo Fisher Scientific, Cat. No. 88860) by In-Fusion cloning (Takara, Cat. No. 639650) to create pCRII-RLuc-IRES-FLuc-A80. A second HindIII restriction site was deleted by site-directed mutagenesis because this enzyme is used for linearization before run-off in vitro transcription. To clone pCRII_SC-LD_SMG6-3xFLAG_A80, the SMG6 coding sequence (CDS) along with a C-terminal 3xFLAG was cloned into pCRII-FL-hRLuc described in (19) by removing the hRLuc sequence and only leaving the SARS-CoV-2 Leader sequence (SC-LD) of the SARS-CoV-2 5΄UTR using InFusion cloning. To obtain pCRII_SC-LD_3xFLAG-HBB-CTE_A80, the beta globin CDS (HBB) with a 70-nucleotide long C-terminal extension (CTE) was cloned into the pCRII_SC-LD_SMG6-3xFLAG_A80, by replacing the SMG6 coding sequence and moving the 3xFLAG tag to the N-terminus using InFusion cloning.
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