Lysis was performed by adding 5 U of DNAse (Fermentas) and 30 g/mL of lysozyme (Sigma) followed by 30 min of incubation on ice and disruption by 10 cycles of sonication

Lysis was performed by adding 5 U of DNAse (Fermentas) and 30 g/mL of lysozyme (Sigma) followed by 30 min of incubation on ice and disruption by 10 cycles of sonication. Effect of SRPIN340 treatment on MAP2K1, MAP2K2, VEGF and FAS expression in HeLa cells. RT-PCR was performed using primers specific for MAP2K1, MAP2K2, VEGF, and FAS genes, and cDNA were derived from HeLa cells after 18 h of treatment with SRPIN340 (100 M). Cells treated with the vehicle DMSO were used like a control. One representative experiment of three is definitely demonstrated. (*) MAP2K1 splicing variant as previously explained [19].(TIF) pone.0134882.s002.tif (82K) GUID:?B7941B06-7FB4-401C-8BFA-5AEDAEEF480B S3 Fig: Superposition of SRPK1 and SRPK2 crystallographic structures. SRPK1 (PDB ID 1WAK, gray) and SRPK2 (PDB ID 2X7G, blue) constructions were aligned attesting their high similarity.(TIF) pone.0134882.s003.tif (676K) GUID:?FD9B9AE9-6047-4D35-962F-E00F8902255C S1 Table: List of primers. (PDF) pone.0134882.s004.pdf (217K) GUID:?5F733431-9796-4720-BADD-58A8AA74FE55 Data Availability StatementAll relevant data are within the paper and its Supporting Info files. Abstract Dysregulation of pre-mRNA splicing machinery activity has been related to the biogenesis of several diseases. The serine/arginine-rich protein kinase family (SRPKs) plays a critical part in regulating pre-mRNA splicing events through the considerable phosphorylation of splicing factors from the family of serine/arginine-rich proteins (SR proteins). Earlier investigations have explained the overexpression of SRPK1 and SRPK2 in leukemia and additional tumor types, suggesting that they would be useful focuses on for developing novel antitumor strategies. Herein, we evaluated the effect of selective pharmacological SRPK inhibition by evaluation of the antileukemia potential of SRPK pharmacological inhibition. In addition, structural data that might clarify SRPIN340s inhibitory activity on SRPK2 will also be described. Experimental Methods Cell lines The leukemia cell lines used were K562 (chronic myelogenous leukemiaCML); KG1 and HL60 (acute myelogenous leukemiaAML); Jurkat, TALL, and Molt4 (T-cell acute lymphoblastic leukemiaCALL-T); and RS4, 697, and Nalm6 (B-cell acute lymphoblastic leukemiaCALL-B). K562, HL60, RS4, and 697 were kindly provided by Dr. Sheila A. Shurtleff (St. Jude Childrens Study Hospital, Memphis, TN). The Nalm6 cell collection was provided by Dr. Angelo Cardoso (Dana-Farber Malignancy Institute, Boston, MA). TALL was kindly provided by Dr. Joao T. Barata (Instituto de Medicina Molecular, Lisboa, Portugal). The KG1, Molt4, and Jurkat cell lines were provided by Dr. Alexandre E. Nowill (Centro Integrado de Pesquisas Oncohematolgicas da Infancia, UNICAMP, Campinas, Brazil). Cells were cultivated in RPMI 1640 (Sigma) medium supplemented with 10% (v/v) fetal bovine serum (FBS) (LGC Biotecnologia), 100 g/mL streptomycin, and 100 devices/mL penicillin at pH 7.2 and 37C less than a 5% CO2 atmosphere. Isolation of PBMC from human being blood Peripheral blood was collected in EDTA tubes, diluted with an equal volume of Hanks balanced salt remedy (HBSS) and combined gently. All methods were performed relating to ethics considerations of the Declaration of Helsinki and were authorized by the ethics committee of the Universidade Federal government de Vi?osa. Later on, samples were layered onto a cushioning of Histopaque 1077 (Sigma) and centrifuged at space temp for 30 min at 400 xto remove insoluble cellular debris. An equal volume of 2X sample buffer comprising 4% (w/v) SDS, 0.2% (w/v) bromophenol blue, 20% (v/v) glycerol, and 100 mM Tris (pH 6.8) was added to the supernatant. Then, the samples were heated to 70C for 10 min. Approximately 1.5×105 cell equivalents were loaded per well of 10% Bis-Tris SDSCpolyacrylamide gel electrophoresis. Later on, proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (GE Healthcare), blocked over night in PBS comprising 5% (w/v) skim.Densitometry analysis of the band intensity was performed using ImageJ software. a calibrator (Ct = Ct(PBMC)Ct(SRPK)). The same approach was attempted with SRPK1, but its manifestation could not be precisely compared with the leukemia cells (observe graph E) because it was barely recognized in the PBMC samples. Nevertheless, this indicates that SRPK1 has very low expression in PBMC, which is in good agreement with our WB assays (Fig 1A) and with previous RT-qPCR reports [23,24]. The primers used in these experiments are detailed in S1 Table.(TIF) pone.0134882.s001.tif (158K) GUID:?032FC8F6-18F5-40A8-835F-9E8A24903E84 S2 Fig: Effect of SRPIN340 treatment on MAP2K1, MAP2K2, VEGF and FAS expression in HeLa cells. RT-PCR was performed using primers specific for MAP2K1, MAP2K2, VEGF, and FAS genes, and cDNA were derived from HeLa cells after 18 h of treatment with SRPIN340 (100 M). Cells treated with the vehicle DMSO were used as a control. One representative experiment of three is usually shown. (*) MAP2K1 splicing variant as previously explained [19].(TIF) pone.0134882.s002.tif (82K) GUID:?B7941B06-7FB4-401C-8BFA-5AEDAEEF480B S3 Fig: Superposition of SRPK1 and SRPK2 crystallographic structures. SRPK1 (PDB ID 1WAK, grey) and SRPK2 (PDB ID 2X7G, blue) structures were aligned attesting their high similarity.(TIF) pone.0134882.s003.tif (676K) GUID:?FD9B9AE9-6047-4D35-962F-E00F8902255C S1 Table: List of primers. (PDF) pone.0134882.s004.pdf (217K) GUID:?5F733431-9796-4720-BADD-58A8AA74FE55 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Dysregulation of pre-mRNA splicing machinery activity has been related to the biogenesis of several diseases. The serine/arginine-rich protein kinase family (SRPKs) plays a critical role in regulating pre-mRNA splicing events through the considerable phosphorylation of splicing factors from the family of serine/arginine-rich proteins (SR proteins). Previous investigations have explained the overexpression of SRPK1 and SRPK2 in leukemia and other cancer types, suggesting that they would be useful targets for developing novel antitumor strategies. Herein, we evaluated the effect of selective pharmacological SRPK inhibition by evaluation of the antileukemia potential of SRPK pharmacological inhibition. In addition, structural data that might explain SRPIN340s inhibitory activity on PRKM10 SRPK2 are also described. Experimental Procedures Cell lines The leukemia cell lines used were K562 (chronic myelogenous leukemiaCML); KG1 and HL60 (acute myelogenous leukemiaAML); Jurkat, TALL, and Molt4 (T-cell acute lymphoblastic leukemiaCALL-T); and RS4, 697, and Nalm6 Rosiglitazone maleate (B-cell acute lymphoblastic leukemiaCALL-B). K562, HL60, RS4, and 697 were kindly provided by Dr. Sheila A. Shurtleff (St. Jude Childrens Research Hospital, Memphis, TN). The Nalm6 cell collection was provided by Dr. Angelo Cardoso (Dana-Farber Malignancy Institute, Boston, MA). TALL was kindly provided by Dr. Joao T. Barata (Instituto de Medicina Molecular, Lisboa, Portugal). The KG1, Molt4, and Jurkat cell lines were provided by Dr. Alexandre E. Nowill (Centro Integrado de Pesquisas Oncohematolgicas da Infancia, UNICAMP, Campinas, Brazil). Cells were cultivated in RPMI 1640 (Sigma) medium supplemented with 10% (v/v) fetal bovine serum (FBS) (LGC Biotecnologia), 100 g/mL streptomycin, and 100 models/mL penicillin at pH 7.2 and 37C under a 5% CO2 atmosphere. Isolation of PBMC from human blood Peripheral blood was collected in EDTA tubes, diluted with an equal volume of Hanks balanced salt answer (HBSS) and mixed gently. All procedures were performed according to ethics considerations of the Declaration of Helsinki and were approved by the ethics committee of the Universidade Federal de Vi?osa. Afterwards, samples were layered onto a cushion of Histopaque 1077 (Sigma) and centrifuged at room heat for 30 min at 400 xto remove insoluble cellular debris. An equal volume of 2X sample buffer made up of 4% (w/v) SDS, 0.2% (w/v) bromophenol blue, 20% (v/v) glycerol, and 100 mM Tris (pH 6.8) was added to the supernatant. Then, the samples were heated to 70C for 10 min. Approximately 1.5×105 cell equivalents were loaded per well of 10% Bis-Tris SDSCpolyacrylamide gel electrophoresis. Afterwards, proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (GE Healthcare), blocked overnight in PBS made up of 5% (w/v) skim milk powder, and then incubated for 2 h with main antibody solutions. Specific kinases were detected using 1:4000 dilutions of anti-SRPK1 and anti-SRPK2 (BD Biosciences). Phosphorylated SR proteins were detected using a 1:1000 dilution of mAb1H4 (Invitrogen) specific for any phospho-epitope common to multiple SR proteins. Each blot was re-probed with a 1:1000 dilution of anti-actin (Sigma), used as an endogenous control in all experiments. Blots were washed in PBS-Tween (PBS-T) and incubated for 2 h in a 1:5000 dilution of a peroxidase-conjugated secondary antibody. Then, proteins were visualized using a Super Transmission Western Pico Chemiluminescent Substrate Package (Thermo Scientific). Cloning, purification and manifestation methods The clone pCMV-SPORT6-SRPK2 was.The values are expressed as the means regular deviation of three independent experiments. graph E) since it was detected in the PBMC examples barely. Nevertheless, this means that that SRPK1 offers very low manifestation in PBMC, which is within good agreement with Rosiglitazone maleate this WB assays (Fig 1A) and with earlier RT-qPCR reviews [23,24]. The primers found in these tests are comprehensive in S1 Desk.(TIF) pone.0134882.s001.tif (158K) GUID:?032FC8F6-18F5-40A8-835F-9E8A24903E84 S2 Fig: Aftereffect of SRPIN340 treatment on MAP2K1, MAP2K2, VEGF and FAS expression in HeLa cells. RT-PCR was performed using primers particular for MAP2K1, MAP2K2, VEGF, and FAS genes, and cDNA had been produced from HeLa cells after 18 h of treatment with SRPIN340 (100 M). Cells treated with the automobile DMSO had been utilized like a control. One representative test of three can be demonstrated. (*) MAP2K1 splicing variant as previously referred to [19].(TIF) pone.0134882.s002.tif (82K) GUID:?B7941B06-7FB4-401C-8BFA-5AEDAEEF480B S3 Fig: Superposition of SRPK1 and SRPK2 crystallographic structures. SRPK1 (PDB Identification 1WAK, gray) and SRPK2 (PDB Identification 2X7G, blue) constructions had been aligned attesting their high similarity.(TIF) pone.0134882.s003.tif (676K) GUID:?FD9B9AE9-6047-4D35-962F-E00F8902255C S1 Desk: Set of primers. (PDF) pone.0134882.s004.pdf (217K) GUID:?5F733431-9796-4720-BADD-58A8AA74FE55 Data Availability StatementAll relevant data are inside the paper and its own Supporting Info files. Abstract Dysregulation of pre-mRNA splicing equipment activity continues to be linked to the biogenesis of many illnesses. The serine/arginine-rich proteins kinase family members (SRPKs) plays a crucial part in regulating pre-mRNA splicing occasions through the intensive phosphorylation of splicing elements from the category of serine/arginine-rich proteins (SR proteins). Earlier investigations have referred to the overexpression of SRPK1 and SRPK2 in leukemia and additional cancer types, recommending that they might be useful focuses on for developing book antitumor strategies. Herein, we examined the result of selective pharmacological SRPK inhibition by evaluation from the antileukemia potential of SRPK pharmacological inhibition. Furthermore, structural data that may clarify SRPIN340s inhibitory activity on SRPK2 will also be described. Experimental Methods Cell lines The leukemia cell lines utilized had been K562 (chronic myelogenous leukemiaCML); KG1 and HL60 (severe myelogenous leukemiaAML); Jurkat, High, and Molt4 (T-cell severe lymphoblastic leukemiaCALL-T); and RS4, 697, and Nalm6 (B-cell severe lymphoblastic leukemiaCALL-B). K562, HL60, RS4, and 697 had been kindly supplied by Dr. Sheila A. Shurtleff (St. Jude Childrens Study Medical center, Memphis, TN). The Nalm6 cell range was supplied by Dr. Angelo Cardoso (Dana-Farber Tumor Institute, Boston, MA). High was kindly supplied by Dr. Joao T. Barata (Instituto de Medicina Molecular, Lisboa, Portugal). The KG1, Molt4, and Jurkat cell lines had been supplied by Dr. Alexandre E. Nowill (Centro Integrado de Pesquisas Oncohematolgicas da Infancia, UNICAMP, Campinas, Brazil). Cells had been cultivated in RPMI 1640 (Sigma) moderate supplemented with 10% (v/v) fetal bovine serum (FBS) (LGC Biotecnologia), 100 g/mL streptomycin, and 100 products/mL penicillin at pH 7.2 and 37C less than a 5% CO2 atmosphere. Isolation of PBMC from human being blood Peripheral bloodstream was gathered in EDTA pipes, diluted with the same level of Hanks well balanced salt option (HBSS) and combined gently. All methods had been performed relating to ethics factors from the Declaration of Helsinki and had been authorized by the ethics committee from the Universidade Federal government de Vi?osa. Later on, examples had been split onto a cushioning of Histopaque 1077 (Sigma) and centrifuged at space temperatures for 30 min at 400 xto remove insoluble mobile debris. The same level of 2X test buffer including 4% (w/v) SDS, 0.2% (w/v) bromophenol blue, 20% (v/v) glycerol, and 100 mM Tris (pH 6.8) was put into the supernatant. After that, the examples had been warmed to 70C for 10 min. Around 1.5×105 cell equivalents were packed per well of 10% Bis-Tris SDSCpolyacrylamide gel electrophoresis. Later on, proteins had been used in a polyvinylidene difluoride (PVDF) membrane (GE Health care), blocked in PBS overnight.(PDF) pone.0134882.s004.pdf (217K) GUID:?5F733431-9796-4720-BADD-58A8AA74FE55 Data Availability StatementAll relevant data are inside the paper and its own Supporting Information documents. Abstract Dysregulation of pre-mRNA splicing equipment activity continues to be linked to the biogenesis of several illnesses. and FAS manifestation in HeLa cells. RT-PCR was performed using primers particular for MAP2K1, MAP2K2, VEGF, and FAS genes, and cDNA had been produced from HeLa cells after 18 h of treatment with SRPIN340 (100 M). Cells treated with the automobile DMSO had been utilized like a control. One representative test of three can be demonstrated. (*) MAP2K1 splicing variant as previously referred to [19].(TIF) pone.0134882.s002.tif (82K) GUID:?B7941B06-7FB4-401C-8BFA-5AEDAEEF480B S3 Fig: Superposition of SRPK1 and SRPK2 crystallographic structures. SRPK1 (PDB Identification 1WAK, gray) and SRPK2 (PDB Identification 2X7G, blue) constructions had been aligned attesting their high similarity.(TIF) pone.0134882.s003.tif (676K) GUID:?FD9B9AE9-6047-4D35-962F-E00F8902255C S1 Desk: List of primers. (PDF) pone.0134882.s004.pdf (217K) GUID:?5F733431-9796-4720-BADD-58A8AA74FE55 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Dysregulation of pre-mRNA splicing machinery activity has been related to the biogenesis of several diseases. The serine/arginine-rich protein kinase family (SRPKs) plays a critical role in regulating pre-mRNA splicing events through the extensive phosphorylation of splicing factors from the family of serine/arginine-rich proteins (SR proteins). Previous investigations have described the overexpression of SRPK1 and SRPK2 in leukemia and other cancer types, suggesting that they would be useful targets for developing novel antitumor strategies. Herein, we evaluated the effect of selective pharmacological SRPK inhibition by evaluation of the antileukemia potential of SRPK pharmacological inhibition. In addition, structural data that might explain SRPIN340s inhibitory activity on SRPK2 are also described. Experimental Procedures Cell lines The leukemia cell lines used were K562 (chronic myelogenous leukemiaCML); KG1 and HL60 (acute myelogenous leukemiaAML); Jurkat, TALL, and Molt4 (T-cell acute lymphoblastic leukemiaCALL-T); and RS4, 697, and Nalm6 (B-cell acute lymphoblastic leukemiaCALL-B). K562, HL60, RS4, and 697 were kindly provided by Dr. Sheila A. Shurtleff (St. Jude Childrens Research Hospital, Rosiglitazone maleate Memphis, TN). The Nalm6 cell line was provided by Dr. Angelo Cardoso (Dana-Farber Cancer Institute, Boston, MA). TALL was kindly provided by Dr. Joao T. Barata (Instituto de Medicina Molecular, Lisboa, Portugal). The KG1, Molt4, and Jurkat cell lines were provided by Dr. Alexandre E. Nowill (Centro Integrado de Pesquisas Oncohematolgicas da Infancia, UNICAMP, Campinas, Brazil). Cells were cultivated in RPMI 1640 (Sigma) medium supplemented with 10% (v/v) fetal bovine serum (FBS) (LGC Biotecnologia), 100 Rosiglitazone maleate g/mL streptomycin, and 100 units/mL penicillin at pH 7.2 and 37C under a 5% CO2 atmosphere. Isolation of PBMC from human blood Peripheral blood was collected in EDTA tubes, diluted with an equal volume of Hanks balanced salt solution (HBSS) and mixed gently. All procedures were performed according to ethics considerations of the Declaration of Helsinki and were approved by the ethics committee of the Universidade Federal de Vi?osa. Afterwards, samples were layered onto a cushion of Histopaque 1077 (Sigma) and centrifuged at room temperature for 30 min at 400 xto remove insoluble cellular debris. An equal volume of 2X sample buffer containing 4% (w/v) SDS, 0.2% (w/v) bromophenol blue, 20% (v/v) glycerol, and 100 mM Tris (pH 6.8) was added to the supernatant. Then, the samples were heated to 70C for 10 min. Approximately 1.5×105 cell equivalents were loaded per well of 10% Bis-Tris SDSCpolyacrylamide gel electrophoresis. Afterwards, proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (GE Healthcare), blocked overnight in PBS containing 5% (w/v) skim milk powder, and then incubated for 2 h with primary antibody solutions. Specific kinases were detected using 1:4000 dilutions of anti-SRPK1 and anti-SRPK2 (BD Biosciences). Phosphorylated SR proteins were detected using a 1:1000 dilution of mAb1H4 (Invitrogen) specific for a phospho-epitope common to multiple SR proteins. Each blot was re-probed with a 1:1000 dilution of anti-actin (Sigma), used as an.A single point charge extended (SPC/E) water model was used to fill up a cubic water box built around previously generated SRPIN340/SPRK2 complexes. The primers used in these experiments are detailed in S1 Table.(TIF) pone.0134882.s001.tif (158K) GUID:?032FC8F6-18F5-40A8-835F-9E8A24903E84 S2 Fig: Effect of SRPIN340 treatment on MAP2K1, MAP2K2, VEGF and FAS expression in HeLa cells. RT-PCR was performed using primers specific for MAP2K1, MAP2K2, VEGF, and FAS genes, and cDNA were derived from HeLa cells after 18 h of treatment with SRPIN340 (100 M). Cells treated with the vehicle DMSO were used as a control. One representative experiment of three is shown. (*) MAP2K1 splicing variant as previously described [19].(TIF) pone.0134882.s002.tif (82K) GUID:?B7941B06-7FB4-401C-8BFA-5AEDAEEF480B S3 Fig: Superposition of SRPK1 and SRPK2 crystallographic structures. SRPK1 (PDB ID 1WAK, grey) and SRPK2 (PDB ID 2X7G, blue) structures were aligned attesting their high similarity.(TIF) pone.0134882.s003.tif (676K) GUID:?FD9B9AE9-6047-4D35-962F-E00F8902255C S1 Desk: Set of primers. (PDF) pone.0134882.s004.pdf (217K) GUID:?5F733431-9796-4720-BADD-58A8AA74FE55 Data Availability StatementAll relevant data are inside the paper and its own Supporting Details files. Abstract Dysregulation of pre-mRNA splicing equipment activity continues to be linked to the biogenesis of many illnesses. The serine/arginine-rich proteins kinase family members (SRPKs) plays a crucial function in regulating pre-mRNA splicing occasions through the comprehensive phosphorylation of splicing elements from the category of serine/arginine-rich proteins (SR proteins). Prior investigations have defined the overexpression of SRPK1 and SRPK2 in leukemia and various other cancer types, recommending that they might be useful goals for developing book antitumor strategies. Herein, we examined the result of selective pharmacological SRPK inhibition by evaluation from the antileukemia potential of SRPK pharmacological inhibition. Furthermore, structural data that may describe SRPIN340s inhibitory activity on SRPK2 may also be described. Experimental Techniques Cell lines The leukemia cell lines utilized had been K562 (chronic myelogenous leukemiaCML); KG1 and HL60 (severe myelogenous leukemiaAML); Jurkat, High, and Molt4 (T-cell severe lymphoblastic leukemiaCALL-T); and RS4, 697, and Nalm6 (B-cell severe lymphoblastic leukemiaCALL-B). K562, HL60, RS4, and 697 had been kindly supplied by Dr. Sheila A. Shurtleff (St. Jude Childrens Analysis Medical center, Memphis, TN). The Nalm6 cell series was supplied by Dr. Angelo Cardoso (Dana-Farber Cancers Institute, Boston, MA). High was kindly supplied by Dr. Joao T. Barata (Instituto de Medicina Molecular, Lisboa, Portugal). The KG1, Molt4, and Jurkat cell lines had been supplied by Dr. Alexandre E. Nowill (Centro Integrado de Pesquisas Oncohematolgicas da Infancia, UNICAMP, Campinas, Brazil). Cells had been cultivated in RPMI 1640 (Sigma) moderate supplemented with 10% (v/v) fetal bovine serum (FBS) (LGC Biotecnologia), 100 g/mL streptomycin, and 100 systems/mL penicillin at pH 7.2 and 37C in a 5% CO2 atmosphere. Isolation of PBMC from individual blood Peripheral bloodstream was gathered in EDTA pipes, diluted with the same level of Hanks well balanced salt alternative (HBSS) and blended gently. All techniques had been performed regarding to ethics factors from the Declaration of Helsinki and had been accepted by the ethics committee from the Universidade Government de Vi?osa. Soon after, samples had been split onto a pillow of Histopaque 1077 (Sigma) and centrifuged at area heat range for 30 min at 400 xto remove Rosiglitazone maleate insoluble mobile debris. The same level of 2X test buffer filled with 4% (w/v) SDS, 0.2% (w/v) bromophenol blue, 20% (v/v) glycerol, and 100 mM Tris (pH 6.8) was put into the supernatant. After that, the samples had been warmed to 70C for 10 min. Around 1.5×105 cell equivalents were packed per well of 10% Bis-Tris SDSCpolyacrylamide gel electrophoresis. Soon after, proteins had been used in a polyvinylidene difluoride (PVDF) membrane (GE Health care), blocked right away in PBS filled with 5% (w/v) skim dairy powder, and incubated for 2 h with principal antibody solutions. Particular kinases had been discovered using 1:4000 dilutions of anti-SRPK1 and anti-SRPK2 (BD Biosciences). Phosphorylated SR protein had been detected utilizing a 1:1000 dilution of mAb1H4 (Invitrogen) particular for the phospho-epitope common to multiple SR protein. Each blot was re-probed using a 1:1000 dilution of anti-actin (Sigma), utilized as an endogenous control in every tests. Blots had been cleaned in PBS-Tween (PBS-T) and incubated for 2 h within a 1:5000 dilution of the peroxidase-conjugated supplementary antibody. Then, protein had been visualized utilizing a Super Indication Western world Pico Chemiluminescent Substrate Package (Thermo Scientific). Cloning, appearance and purification techniques The clone pCMV-SPORT6-SRPK2 was bought in the Mammalian Gene Collection (Invitrogen). This clone allowed amplification of full-length SRPK2 cDNA by PCR and subcloning in to the pET28a-HIS-TEV vector [33], a improved.