Recombinant:Article Title: Immediate early splicing controls translation in activated T-cells and is mediated by hnRNPC2 phosphorylation.
Article Snippet: .. Reagents and tools table Reagent/resource Reference or source Identifier or catalog number Experimental models Top10 competent E. coli This study N/A BL21 RIL E. coli Novagen 70954 Jurkat Kristen Lynch, UPenn N/A Primary mouse T cells This study N/A EL4 ATCC, USA TIB-39TM HEK293 ATCC, USA CRL-1573TM HeLa ATCC, USA CCL-2TM RAW264.7 ATCC, USA TIB-71TM N2a MDC, Berlin N/A Recombinant DNA pCDNA Thermo Fisher Scientific V79020 TwinStrep-SUMO tagged hnRNPC2 construct Thermo Fisher Scientific pBS-PKCθ Mischak et al, 1993 #8426 pFLAG-N3-PKCθ This study N/A pTWIST CMV_EIF5A_WT Twist Bioscience N/A pTWIST CMV_EIF5A_IR Twist Bioscience N/A pFLAG-N3-hnRNPC2_WT This study N/A pFLAG-N3- hnRNPC2_S115D This study N/A pFLAG-N3- hnRNPC2_S→A/Y This study N/A pSpCas9(BB)-2A-GFP (pX458) Addgene #48138 Antibodies (dilution) hnRNP C1/C2 (1:1000) Santa Cruz Biotechnology sc-32308 p-ERK (1:1000) Santa Cruz Biotechnology sc-7383 ERK 1⁄2 (1:1000) Santa Cruz Biotechnology sc-514302 DYKDDDDK Tag (1:2000) Cell Signaling Technology #14793 GAPDH (1:2000) Santa Cruz Biotechnology sc-32233 hnRNPL (1:2000) Santa Cruz Biotechnology sc-32317 puromycin (1:5000) Sigma-Aldrich 12D10 anti-mouse IgG HRP, (1:5000) Cell Signaling Technology #7076s anti-rabbit IgG HRP, (1:5000) Cell Signaling Technology #7074s Oligonucleotides and other sequence-based reagents Anti-sense morpholinos Gene Tools Table 1 siRNA sequences Eurofins Scientific Table 2 gRNAs #1 5’-CTCTAC TCAGGTCCGGAAC Eurofins Scientific N/A #2: 5’ -CTGCATTGT GTCCATCAGT Eurofins Scientific N/A RT-PCR primers Eurofins Scientific Table 3 RNA sequences Eurofins Scientific Table 4 Reagent/resource Reference or source Identifier or catalog number Chemicals, enzymes and other reagents DMEM High Glucose Biowest L0104 Trypsin Gibco 25200-056 RPMI 1640 Biowest L0500 Phorbol 12-myristate 13- acetate Sigma-Aldrich P8139 anti-CD3 antibody (1 μg/ml) BioLegend OKT3 Lipopolysaccharide (LPS) Thermo Fisher Scientific 00-4976-03 FastAP Thermosensitive Alkaline Phosphatase Thermo Fisher Scientific EF0654 Potassium chloride Carl Roth. .. Germany 6781.1 cycloheximide Sigma-Aldrich 239763-M PD0325901 (MEK1/2 inhibitor) New England Biolabs 9903S MK2206 (AKT1/2/3 inhibitor) Biomol SYN-1162M001 VX-745 (p38 inhibitor) Biomol Cay18075-1 Ruxolitinib (JAK1/2 inhibitor) Biomol Cay11609-1 CD28 Thermo Fisher Scientific AB_468926 PKCθ inhibitor Selleck S6577 DNase I Biozym 170500 ROTIFect Carl Roth, Germany P001.1 Lipofectamine 2000 Thermo Fisher Scientific 11668019 Opti-MEM (Opti-MEM® I (1X) + GlutaMAXTM-I - Reduced Serum Medium Thermo Fisher Scientific 51985091 PBS Biowest L0615-500 BpiI Life Technologies FD1014 puromycin (1 μg/ml) InvivoGen ant-pr-1 proteinase K (20mg/mL) Serva 33755.01 The Quick-Load® 1 kb Plus DNA Ladder New England Biolabs Inc. N0550S ROTI®Aqua-P/C/I Carl Roth X985.1 RNA-se inhibitor, RiboLock Life Technologies EO0382 MMuLV reverse transcriptase Enzymatics Inc. 280550 T4 PNK (10 U/ml) Life Technologies EK0032 γ-32P-dATP (10 μCI/μl, 6000 Ci/mmol, Hartmann Analytic SRP-501 Absolute QPCR SYBR Green Mix Biozym 331416 XhoI Life Technologies FD00695 NheI Life Technologies FD0974 PMSF -1:100 Carl Roth 6367.2 Aprotinin-1:200 Carl Roth A162.3 Leupeptin-1:200 Carl Roth CN33.3 Vanadate-1:500 Sigma-Aldrich S6508-50G Coomassie Brilliant Blue G250 Carl Roth 9598.1 © The Author(s) The EMBO Journal 15 D ow nloaded from https://w w w .em bopress.org on February 19, 2025 from IP 180.244.138.216.
Construct:Article Title: Immediate early splicing controls translation in activated T-cells and is mediated by hnRNPC2 phosphorylation.
Article Snippet: .. Reagents and tools table Reagent/resource Reference or source Identifier or catalog number Experimental models Top10 competent E. coli This study N/A BL21 RIL E. coli Novagen 70954 Jurkat Kristen Lynch, UPenn N/A Primary mouse T cells This study N/A EL4 ATCC, USA TIB-39TM HEK293 ATCC, USA CRL-1573TM HeLa ATCC, USA CCL-2TM RAW264.7 ATCC, USA TIB-71TM N2a MDC, Berlin N/A Recombinant DNA pCDNA Thermo Fisher Scientific V79020 TwinStrep-SUMO tagged hnRNPC2 construct Thermo Fisher Scientific pBS-PKCθ Mischak et al, 1993 #8426 pFLAG-N3-PKCθ This study N/A pTWIST CMV_EIF5A_WT Twist Bioscience N/A pTWIST CMV_EIF5A_IR Twist Bioscience N/A pFLAG-N3-hnRNPC2_WT This study N/A pFLAG-N3- hnRNPC2_S115D This study N/A pFLAG-N3- hnRNPC2_S→A/Y This study N/A pSpCas9(BB)-2A-GFP (pX458) Addgene #48138 Antibodies (dilution) hnRNP C1/C2 (1:1000) Santa Cruz Biotechnology sc-32308 p-ERK (1:1000) Santa Cruz Biotechnology sc-7383 ERK 1⁄2 (1:1000) Santa Cruz Biotechnology sc-514302 DYKDDDDK Tag (1:2000) Cell Signaling Technology #14793 GAPDH (1:2000) Santa Cruz Biotechnology sc-32233 hnRNPL (1:2000) Santa Cruz Biotechnology sc-32317 puromycin (1:5000) Sigma-Aldrich 12D10 anti-mouse IgG HRP, (1:5000) Cell Signaling Technology #7076s anti-rabbit IgG HRP, (1:5000) Cell Signaling Technology #7074s Oligonucleotides and other sequence-based reagents Anti-sense morpholinos Gene Tools Table 1 siRNA sequences Eurofins Scientific Table 2 gRNAs #1 5’-CTCTAC TCAGGTCCGGAAC Eurofins Scientific N/A #2: 5’ -CTGCATTGT GTCCATCAGT Eurofins Scientific N/A RT-PCR primers Eurofins Scientific Table 3 RNA sequences Eurofins Scientific Table 4 Reagent/resource Reference or source Identifier or catalog number Chemicals, enzymes and other reagents DMEM High Glucose Biowest L0104 Trypsin Gibco 25200-056 RPMI 1640 Biowest L0500 Phorbol 12-myristate 13- acetate Sigma-Aldrich P8139 anti-CD3 antibody (1 μg/ml) BioLegend OKT3 Lipopolysaccharide (LPS) Thermo Fisher Scientific 00-4976-03 FastAP Thermosensitive Alkaline Phosphatase Thermo Fisher Scientific EF0654 Potassium chloride Carl Roth. .. Germany 6781.1 cycloheximide Sigma-Aldrich 239763-M PD0325901 (MEK1/2 inhibitor) New England Biolabs 9903S MK2206 (AKT1/2/3 inhibitor) Biomol SYN-1162M001 VX-745 (p38 inhibitor) Biomol Cay18075-1 Ruxolitinib (JAK1/2 inhibitor) Biomol Cay11609-1 CD28 Thermo Fisher Scientific AB_468926 PKCθ inhibitor Selleck S6577 DNase I Biozym 170500 ROTIFect Carl Roth, Germany P001.1 Lipofectamine 2000 Thermo Fisher Scientific 11668019 Opti-MEM (Opti-MEM® I (1X) + GlutaMAXTM-I - Reduced Serum Medium Thermo Fisher Scientific 51985091 PBS Biowest L0615-500 BpiI Life Technologies FD1014 puromycin (1 μg/ml) InvivoGen ant-pr-1 proteinase K (20mg/mL) Serva 33755.01 The Quick-Load® 1 kb Plus DNA Ladder New England Biolabs Inc. N0550S ROTI®Aqua-P/C/I Carl Roth X985.1 RNA-se inhibitor, RiboLock Life Technologies EO0382 MMuLV reverse transcriptase Enzymatics Inc. 280550 T4 PNK (10 U/ml) Life Technologies EK0032 γ-32P-dATP (10 μCI/μl, 6000 Ci/mmol, Hartmann Analytic SRP-501 Absolute QPCR SYBR Green Mix Biozym 331416 XhoI Life Technologies FD00695 NheI Life Technologies FD0974 PMSF -1:100 Carl Roth 6367.2 Aprotinin-1:200 Carl Roth A162.3 Leupeptin-1:200 Carl Roth CN33.3 Vanadate-1:500 Sigma-Aldrich S6508-50G Coomassie Brilliant Blue G250 Carl Roth 9598.1 © The Author(s) The EMBO Journal 15 D ow nloaded from https://w w w .em bopress.org on February 19, 2025 from IP 180.244.138.216.
Sequencing:Article Title: Immediate early splicing controls translation in activated T-cells and is mediated by hnRNPC2 phosphorylation.
Article Snippet: .. Reagents and tools table Reagent/resource Reference or source Identifier or catalog number Experimental models Top10 competent E. coli This study N/A BL21 RIL E. coli Novagen 70954 Jurkat Kristen Lynch, UPenn N/A Primary mouse T cells This study N/A EL4 ATCC, USA TIB-39TM HEK293 ATCC, USA CRL-1573TM HeLa ATCC, USA CCL-2TM RAW264.7 ATCC, USA TIB-71TM N2a MDC, Berlin N/A Recombinant DNA pCDNA Thermo Fisher Scientific V79020 TwinStrep-SUMO tagged hnRNPC2 construct Thermo Fisher Scientific pBS-PKCθ Mischak et al, 1993 #8426 pFLAG-N3-PKCθ This study N/A pTWIST CMV_EIF5A_WT Twist Bioscience N/A pTWIST CMV_EIF5A_IR Twist Bioscience N/A pFLAG-N3-hnRNPC2_WT This study N/A pFLAG-N3- hnRNPC2_S115D This study N/A pFLAG-N3- hnRNPC2_S→A/Y This study N/A pSpCas9(BB)-2A-GFP (pX458) Addgene #48138 Antibodies (dilution) hnRNP C1/C2 (1:1000) Santa Cruz Biotechnology sc-32308 p-ERK (1:1000) Santa Cruz Biotechnology sc-7383 ERK 1⁄2 (1:1000) Santa Cruz Biotechnology sc-514302 DYKDDDDK Tag (1:2000) Cell Signaling Technology #14793 GAPDH (1:2000) Santa Cruz Biotechnology sc-32233 hnRNPL (1:2000) Santa Cruz Biotechnology sc-32317 puromycin (1:5000) Sigma-Aldrich 12D10 anti-mouse IgG HRP, (1:5000) Cell Signaling Technology #7076s anti-rabbit IgG HRP, (1:5000) Cell Signaling Technology #7074s Oligonucleotides and other sequence-based reagents Anti-sense morpholinos Gene Tools Table 1 siRNA sequences Eurofins Scientific Table 2 gRNAs #1 5’-CTCTAC TCAGGTCCGGAAC Eurofins Scientific N/A #2: 5’ -CTGCATTGT GTCCATCAGT Eurofins Scientific N/A RT-PCR primers Eurofins Scientific Table 3 RNA sequences Eurofins Scientific Table 4 Reagent/resource Reference or source Identifier or catalog number Chemicals, enzymes and other reagents DMEM High Glucose Biowest L0104 Trypsin Gibco 25200-056 RPMI 1640 Biowest L0500 Phorbol 12-myristate 13- acetate Sigma-Aldrich P8139 anti-CD3 antibody (1 μg/ml) BioLegend OKT3 Lipopolysaccharide (LPS) Thermo Fisher Scientific 00-4976-03 FastAP Thermosensitive Alkaline Phosphatase Thermo Fisher Scientific EF0654 Potassium chloride Carl Roth. .. Germany 6781.1 cycloheximide Sigma-Aldrich 239763-M PD0325901 (MEK1/2 inhibitor) New England Biolabs 9903S MK2206 (AKT1/2/3 inhibitor) Biomol SYN-1162M001 VX-745 (p38 inhibitor) Biomol Cay18075-1 Ruxolitinib (JAK1/2 inhibitor) Biomol Cay11609-1 CD28 Thermo Fisher Scientific AB_468926 PKCθ inhibitor Selleck S6577 DNase I Biozym 170500 ROTIFect Carl Roth, Germany P001.1 Lipofectamine 2000 Thermo Fisher Scientific 11668019 Opti-MEM (Opti-MEM® I (1X) + GlutaMAXTM-I - Reduced Serum Medium Thermo Fisher Scientific 51985091 PBS Biowest L0615-500 BpiI Life Technologies FD1014 puromycin (1 μg/ml) InvivoGen ant-pr-1 proteinase K (20mg/mL) Serva 33755.01 The Quick-Load® 1 kb Plus DNA Ladder New England Biolabs Inc. N0550S ROTI®Aqua-P/C/I Carl Roth X985.1 RNA-se inhibitor, RiboLock Life Technologies EO0382 MMuLV reverse transcriptase Enzymatics Inc. 280550 T4 PNK (10 U/ml) Life Technologies EK0032 γ-32P-dATP (10 μCI/μl, 6000 Ci/mmol, Hartmann Analytic SRP-501 Absolute QPCR SYBR Green Mix Biozym 331416 XhoI Life Technologies FD00695 NheI Life Technologies FD0974 PMSF -1:100 Carl Roth 6367.2 Aprotinin-1:200 Carl Roth A162.3 Leupeptin-1:200 Carl Roth CN33.3 Vanadate-1:500 Sigma-Aldrich S6508-50G Coomassie Brilliant Blue G250 Carl Roth 9598.1 © The Author(s) The EMBO Journal 15 D ow nloaded from https://w w w .em bopress.org on February 19, 2025 from IP 180.244.138.216.
Reverse Transcription Polymerase Chain Reaction:Article Title: Immediate early splicing controls translation in activated T-cells and is mediated by hnRNPC2 phosphorylation.
Article Snippet: .. Reagents and tools table Reagent/resource Reference or source Identifier or catalog number Experimental models Top10 competent E. coli This study N/A BL21 RIL E. coli Novagen 70954 Jurkat Kristen Lynch, UPenn N/A Primary mouse T cells This study N/A EL4 ATCC, USA TIB-39TM HEK293 ATCC, USA CRL-1573TM HeLa ATCC, USA CCL-2TM RAW264.7 ATCC, USA TIB-71TM N2a MDC, Berlin N/A Recombinant DNA pCDNA Thermo Fisher Scientific V79020 TwinStrep-SUMO tagged hnRNPC2 construct Thermo Fisher Scientific pBS-PKCθ Mischak et al, 1993 #8426 pFLAG-N3-PKCθ This study N/A pTWIST CMV_EIF5A_WT Twist Bioscience N/A pTWIST CMV_EIF5A_IR Twist Bioscience N/A pFLAG-N3-hnRNPC2_WT This study N/A pFLAG-N3- hnRNPC2_S115D This study N/A pFLAG-N3- hnRNPC2_S→A/Y This study N/A pSpCas9(BB)-2A-GFP (pX458) Addgene #48138 Antibodies (dilution) hnRNP C1/C2 (1:1000) Santa Cruz Biotechnology sc-32308 p-ERK (1:1000) Santa Cruz Biotechnology sc-7383 ERK 1⁄2 (1:1000) Santa Cruz Biotechnology sc-514302 DYKDDDDK Tag (1:2000) Cell Signaling Technology #14793 GAPDH (1:2000) Santa Cruz Biotechnology sc-32233 hnRNPL (1:2000) Santa Cruz Biotechnology sc-32317 puromycin (1:5000) Sigma-Aldrich 12D10 anti-mouse IgG HRP, (1:5000) Cell Signaling Technology #7076s anti-rabbit IgG HRP, (1:5000) Cell Signaling Technology #7074s Oligonucleotides and other sequence-based reagents Anti-sense morpholinos Gene Tools Table 1 siRNA sequences Eurofins Scientific Table 2 gRNAs #1 5’-CTCTAC TCAGGTCCGGAAC Eurofins Scientific N/A #2: 5’ -CTGCATTGT GTCCATCAGT Eurofins Scientific N/A RT-PCR primers Eurofins Scientific Table 3 RNA sequences Eurofins Scientific Table 4 Reagent/resource Reference or source Identifier or catalog number Chemicals, enzymes and other reagents DMEM High Glucose Biowest L0104 Trypsin Gibco 25200-056 RPMI 1640 Biowest L0500 Phorbol 12-myristate 13- acetate Sigma-Aldrich P8139 anti-CD3 antibody (1 μg/ml) BioLegend OKT3 Lipopolysaccharide (LPS) Thermo Fisher Scientific 00-4976-03 FastAP Thermosensitive Alkaline Phosphatase Thermo Fisher Scientific EF0654 Potassium chloride Carl Roth. .. Germany 6781.1 cycloheximide Sigma-Aldrich 239763-M PD0325901 (MEK1/2 inhibitor) New England Biolabs 9903S MK2206 (AKT1/2/3 inhibitor) Biomol SYN-1162M001 VX-745 (p38 inhibitor) Biomol Cay18075-1 Ruxolitinib (JAK1/2 inhibitor) Biomol Cay11609-1 CD28 Thermo Fisher Scientific AB_468926 PKCθ inhibitor Selleck S6577 DNase I Biozym 170500 ROTIFect Carl Roth, Germany P001.1 Lipofectamine 2000 Thermo Fisher Scientific 11668019 Opti-MEM (Opti-MEM® I (1X) + GlutaMAXTM-I - Reduced Serum Medium Thermo Fisher Scientific 51985091 PBS Biowest L0615-500 BpiI Life Technologies FD1014 puromycin (1 μg/ml) InvivoGen ant-pr-1 proteinase K (20mg/mL) Serva 33755.01 The Quick-Load® 1 kb Plus DNA Ladder New England Biolabs Inc. N0550S ROTI®Aqua-P/C/I Carl Roth X985.1 RNA-se inhibitor, RiboLock Life Technologies EO0382 MMuLV reverse transcriptase Enzymatics Inc. 280550 T4 PNK (10 U/ml) Life Technologies EK0032 γ-32P-dATP (10 μCI/μl, 6000 Ci/mmol, Hartmann Analytic SRP-501 Absolute QPCR SYBR Green Mix Biozym 331416 XhoI Life Technologies FD00695 NheI Life Technologies FD0974 PMSF -1:100 Carl Roth 6367.2 Aprotinin-1:200 Carl Roth A162.3 Leupeptin-1:200 Carl Roth CN33.3 Vanadate-1:500 Sigma-Aldrich S6508-50G Coomassie Brilliant Blue G250 Carl Roth 9598.1 © The Author(s) The EMBO Journal 15 D ow nloaded from https://w w w .em bopress.org on February 19, 2025 from IP 180.244.138.216.
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