Journal: Nature Communications

Article Title: PRKCSH contributes to tumorigenesis by selective boosting of IRE1 signaling pathway

doi: 10.1038/s41467-019-11019-w

Figure Lengend Snippet: Internal E/P domain of PRKCSH is required for its interaction with IRE1α and boosting IRE1α activation under ER stress. a Immunoblot analysis of association between endogenous PRKCSH and IRE1α using immunoprecipitates from L02 cells treated with 10 μg/mL TM (left) or glucose-free medium (middle) for the indicated time, and from Huh-7 cells treated with 10 μg/mL TM (right) for the indicated time. b Immunoblot analysis of complex formation between endogenous IRE1α and Flag-tagged PRKCSH immunoprecipitated from transfected L02 cells treated with 10 μg/mL TM for the indicated time. Immunoprecipitation was performed by using anti-IRE1α antibody and normal rabbit IgG as a control antibody. c Immunoblot analysis of complex formation between endogenous PRKCSH and Flag-tagged IRE1α. Immunoprecipitates were prepared from transfected L02 cells treated with 10 μg/mL TM for the indicated time. Immunoprecipitation was performed by using anti-PRKCSH antibody and normal rabbit IgG. d Schematic diagram of the functional domains of PRKCSH and its recombinant variants (left part). Immunoblot analysis of in vitro complex formation between IRE1α and GST-tagged PRKCSH protein (right part). e , f Schematic presentation of wild-type and deletion mutants of PRKCSH (upper part). Immunoblot analysis of complex formation between endogenous IRE1α and Flag-tagged mutant PRKCSHs (lower part). Immunoprecipitates were prepared from transfected L02 cells treated with 10 μg/mL TM for 1 h. Immunocytochemical analysis of L02 cells transfected with Flag-tagged ΔG2B and ΔS/G2B mutant PRKCSH ( f , middle). Calnexin was used as an ER marker. Scale bars represent 5 µm. g Immunoblot analysis of association between endogenous PRKCSH and GIIα. Immunoprecipitates were prepared from L02 (top) or Huh-7 cells (bottom) treated with 10 μg/mL TM for the indicated time. Immunoprecipitation was performed by using anti-GIIα antibody and normal rabbit IgG. h Model of PRKCSH complex formation. Under resting conditions, PRKCSH associates with the GIIα subunit via the G2B domain; this domain inhibits PRKCSH interaction with IRE1α. Under ER stress, PRKCSH dissociates from GIIα and then binds to IRE1α via the E/P domain

Article Snippet: For immunocytochemistry and immunohistochemistry, we used following antibodies: PRKCSH (Santa Cruz Biotechnology, Cat:sc-46685, 1:200), Calnexin (Cell Signaling, Cat:2679, 1:50), 20S Proteasome α4 (Santa Cruz Biotechnology, Cat:sc-271297, 1:200), Alexa Fluor 546 anti-mouse IgG (ThermoFisher Scientific, Cat:A11030, 1:500), and Alexa Fluor 488 anti-rabbit IgG (ThermoFisher Scientific, Cat:A27034, 1:500). and normal rabbit IgG (2729) were obtained from Cell Signaling Technology.

Techniques: Activation Assay, Western Blot, Immunoprecipitation, Transfection, Control, Functional Assay, Recombinant, In Vitro, Mutagenesis, Marker

Journal: Nature protocols

Article Title: Compartmentalized partnered replication for the directed evolution of genetic parts and circuits

doi: 10.1038/nprot.2017.119

Figure Lengend Snippet: General CPR concept, (a) Schematic of CPR principle. A gene circuit is generated in which a partner-gene activity allows the expression of a DNA polymerase in bacterial cells. Inactive gene variants lead to no expression of the DNA polymerase. The genetic circuit containing the diversified partner-gene pool is expressed in vivo, allowing DNA polymerase production only in cells with active partner-gene variants. The live cells are subsequently emulsified to produce no more than a single cell per emulsion droplet. The initial boiling step of the ePCR lyses the cells, releasing the produced DNA polymerase protein as well as the partner-gene-encoding plasmid into the aqueous solution of the emulsion droplet. Ensuing thermal cycling amplifies only the active partner-gene variant, which is recovered and used in the next round of CPR selection, (b) Examples of genetic circuits for CPR. Partner-gene function can be linked to expression of a DNA polymerase in a number of ways, as is demonstrated by the example of T7 RNAP, pol, polymerase; tRNA synthetase, and tRNA engineering.

Article Snippet: 5Prime Phase Lock Gel Heavy tubes (QuantaBio, cat. no. 2302830) Restriction enzyme Dpnl (NEB, cat. no. R0176) CutSmart Buffer, 10× (NEB, cat. no. B7204) QIAquick PCR Purification Kit (Qiagen, cat. no. 28104) Sodium acetate (Sigma-Aldrich, cat. no. S2889) Glacial acetic acid (Fisher, cat. no. A38) Recovery PCR list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 Phusion high-fidelity DNA polymerase (supplied with 5× HF buffer; NEB, cat. no. M0530) PCR primers (Integrated DNA Technologies; sequences listed in ) table ft1 table-wrap mode="anchored" t5 TABLE 2 | caption a7 Primer Sequence 5’−3’ Purpose Rec-For B (AJM.15) GATACAATACAACCAG CGATAGACTCAGG Recovery T7 RNAP Rec-Rev B (AJM.16) CAATACCATACTTTTCA TGTGCCCAGACCACTG Recovery T7 RNAP Rec-For A (JWE.46) CCAAATCAGAAAGAA ACTCAACATCGTCAC Recovery tRNA Rec-Rev A (JWE.47) CGTCTACCCGAAGCCTC TACTATGATCAGT Recovery tRNA Open in a separate window Primers for Recovery PCR. dNTP mix, 10 mM each (Thermo Fisher, cat. no. R0192) TAE, 50× liquid concentrate (Apex Bioresearch Products, cat. no. 20–194) Seakem LE Agarose (Lonza, cat. no. 50004) Ethidium bromide (Thermo Fisher, cat. no. 15585011) !

Techniques: Generated, Activity Assay, Expressing, In Vivo, Produced, Plasmid Preparation, Variant Assay, Selection

Journal: Nature protocols

Article Title: Compartmentalized partnered replication for the directed evolution of genetic parts and circuits

doi: 10.1038/nprot.2017.119

Figure Lengend Snippet: Overview and time line of experiments. A CPR procedure encompasses bacterial expression of the DNA polymerase and partner genes for several hours, emulsification of the bacterial cells in water-oil droplets, in vitro amplification of active partner genes via ePCR, breaking of the emulsions, purification of amplified DNA from cell debris and PCR primers, purification of amplified DNA from background plasmid DNA by Dpnl digestion, and final recovery and bulk amplification of the partner gene using an additional recovery PCR. A single round of CPR typically takes 3–5 d, and the procedure can be repeated 3–10 times until no more enrichment is observed.

Article Snippet: 5Prime Phase Lock Gel Heavy tubes (QuantaBio, cat. no. 2302830) Restriction enzyme Dpnl (NEB, cat. no. R0176) CutSmart Buffer, 10× (NEB, cat. no. B7204) QIAquick PCR Purification Kit (Qiagen, cat. no. 28104) Sodium acetate (Sigma-Aldrich, cat. no. S2889) Glacial acetic acid (Fisher, cat. no. A38) Recovery PCR list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 Phusion high-fidelity DNA polymerase (supplied with 5× HF buffer; NEB, cat. no. M0530) PCR primers (Integrated DNA Technologies; sequences listed in ) table ft1 table-wrap mode="anchored" t5 TABLE 2 | caption a7 Primer Sequence 5’−3’ Purpose Rec-For B (AJM.15) GATACAATACAACCAG CGATAGACTCAGG Recovery T7 RNAP Rec-Rev B (AJM.16) CAATACCATACTTTTCA TGTGCCCAGACCACTG Recovery T7 RNAP Rec-For A (JWE.46) CCAAATCAGAAAGAA ACTCAACATCGTCAC Recovery tRNA Rec-Rev A (JWE.47) CGTCTACCCGAAGCCTC TACTATGATCAGT Recovery tRNA Open in a separate window Primers for Recovery PCR. dNTP mix, 10 mM each (Thermo Fisher, cat. no. R0192) TAE, 50× liquid concentrate (Apex Bioresearch Products, cat. no. 20–194) Seakem LE Agarose (Lonza, cat. no. 50004) Ethidium bromide (Thermo Fisher, cat. no. 15585011) !

Techniques: Expressing, In Vitro, Amplification, Purification, Plasmid Preparation

Journal: Nature protocols

Article Title: Compartmentalized partnered replication for the directed evolution of genetic parts and circuits

doi: 10.1038/nprot.2017.119

Figure Lengend Snippet: Troubleshooting table.

Article Snippet: 5Prime Phase Lock Gel Heavy tubes (QuantaBio, cat. no. 2302830) Restriction enzyme Dpnl (NEB, cat. no. R0176) CutSmart Buffer, 10× (NEB, cat. no. B7204) QIAquick PCR Purification Kit (Qiagen, cat. no. 28104) Sodium acetate (Sigma-Aldrich, cat. no. S2889) Glacial acetic acid (Fisher, cat. no. A38) Recovery PCR list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 Phusion high-fidelity DNA polymerase (supplied with 5× HF buffer; NEB, cat. no. M0530) PCR primers (Integrated DNA Technologies; sequences listed in ) table ft1 table-wrap mode="anchored" t5 TABLE 2 | caption a7 Primer Sequence 5’−3’ Purpose Rec-For B (AJM.15) GATACAATACAACCAG CGATAGACTCAGG Recovery T7 RNAP Rec-Rev B (AJM.16) CAATACCATACTTTTCA TGTGCCCAGACCACTG Recovery T7 RNAP Rec-For A (JWE.46) CCAAATCAGAAAGAA ACTCAACATCGTCAC Recovery tRNA Rec-Rev A (JWE.47) CGTCTACCCGAAGCCTC TACTATGATCAGT Recovery tRNA Open in a separate window Primers for Recovery PCR. dNTP mix, 10 mM each (Thermo Fisher, cat. no. R0192) TAE, 50× liquid concentrate (Apex Bioresearch Products, cat. no. 20–194) Seakem LE Agarose (Lonza, cat. no. 50004) Ethidium bromide (Thermo Fisher, cat. no. 15585011) !

Techniques: Growth Assay, Concentration Assay, Positive Control, Western Blot, Sequencing, Expressing, Amplification, Plasmid Preparation, Selection, Variant Assay, Functional Assay

Journal: Nature protocols

Article Title: Compartmentalized partnered replication for the directed evolution of genetic parts and circuits

doi: 10.1038/nprot.2017.119

Figure Lengend Snippet:

Article Snippet: 5Prime Phase Lock Gel Heavy tubes (QuantaBio, cat. no. 2302830) Restriction enzyme Dpnl (NEB, cat. no. R0176) CutSmart Buffer, 10× (NEB, cat. no. B7204) QIAquick PCR Purification Kit (Qiagen, cat. no. 28104) Sodium acetate (Sigma-Aldrich, cat. no. S2889) Glacial acetic acid (Fisher, cat. no. A38) Recovery PCR list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 Phusion high-fidelity DNA polymerase (supplied with 5× HF buffer; NEB, cat. no. M0530) PCR primers (Integrated DNA Technologies; sequences listed in ) table ft1 table-wrap mode="anchored" t5 TABLE 2 | caption a7 Primer Sequence 5’−3’ Purpose Rec-For B (AJM.15) GATACAATACAACCAG CGATAGACTCAGG Recovery T7 RNAP Rec-Rev B (AJM.16) CAATACCATACTTTTCA TGTGCCCAGACCACTG Recovery T7 RNAP Rec-For A (JWE.46) CCAAATCAGAAAGAA ACTCAACATCGTCAC Recovery tRNA Rec-Rev A (JWE.47) CGTCTACCCGAAGCCTC TACTATGATCAGT Recovery tRNA Open in a separate window Primers for Recovery PCR. dNTP mix, 10 mM each (Thermo Fisher, cat. no. R0192) TAE, 50× liquid concentrate (Apex Bioresearch Products, cat. no. 20–194) Seakem LE Agarose (Lonza, cat. no. 50004) Ethidium bromide (Thermo Fisher, cat. no. 15585011) !

Techniques: Concentration Assay

Journal: Nature protocols

Article Title: Compartmentalized partnered replication for the directed evolution of genetic parts and circuits

doi: 10.1038/nprot.2017.119

Figure Lengend Snippet:

Article Snippet: 5Prime Phase Lock Gel Heavy tubes (QuantaBio, cat. no. 2302830) Restriction enzyme Dpnl (NEB, cat. no. R0176) CutSmart Buffer, 10× (NEB, cat. no. B7204) QIAquick PCR Purification Kit (Qiagen, cat. no. 28104) Sodium acetate (Sigma-Aldrich, cat. no. S2889) Glacial acetic acid (Fisher, cat. no. A38) Recovery PCR list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 Phusion high-fidelity DNA polymerase (supplied with 5× HF buffer; NEB, cat. no. M0530) PCR primers (Integrated DNA Technologies; sequences listed in ) table ft1 table-wrap mode="anchored" t5 TABLE 2 | caption a7 Primer Sequence 5’−3’ Purpose Rec-For B (AJM.15) GATACAATACAACCAG CGATAGACTCAGG Recovery T7 RNAP Rec-Rev B (AJM.16) CAATACCATACTTTTCA TGTGCCCAGACCACTG Recovery T7 RNAP Rec-For A (JWE.46) CCAAATCAGAAAGAA ACTCAACATCGTCAC Recovery tRNA Rec-Rev A (JWE.47) CGTCTACCCGAAGCCTC TACTATGATCAGT Recovery tRNA Open in a separate window Primers for Recovery PCR. dNTP mix, 10 mM each (Thermo Fisher, cat. no. R0192) TAE, 50× liquid concentrate (Apex Bioresearch Products, cat. no. 20–194) Seakem LE Agarose (Lonza, cat. no. 50004) Ethidium bromide (Thermo Fisher, cat. no. 15585011) !

Techniques: Concentration Assay