flag tagged prmt5 recombinant protein  (Sino Biological)

Journal: Oncogene

Article Title: PRMT5 methylating SMAD4 activates TGF-β signaling and promotes colorectal cancer metastasis.

doi: 10.1038/s41388-023-02674-x

Figure Lengend Snippet: Fig. 5 Targeting the methylation of SMAD4 at R361 inhibits CRC metastasis in vivo. A, B 2 × 106 luciferase-expressing LoVo cells of different genotypes, shNC, PRMT5 sh#1 and sh#2, were injected intrasplenic to BALB/c nude mice. After 6 weeks, bioluminescence was detected, then livers were dissected, and metastatic tumors were weighed. Representative images (A) and quantitative analysis of metastatic tumor weight (B) (*p < 0.05, **p < 0.01, t-test). C IHC staining of PRMT5 and SMAD4 R361me2s in tumor tissue. D, E 2 × 106 luciferase-expressing cells of different genotypes, WT, R361K mutant, PRMT5 overexpressing and PRMT5 overexpressing R361K mutant, were injected intrasplenic into BALB/c nude mice. After 3 weeks of tumor cell injection, half of the mice injected with WT and R361K cells were treated with GSK3326595 (40 mg/kg) every 3 days. After another 3 weeks, bioluminescence was detected, then livers were dissected, and metastatic tumors were weighed. Representative images (D) and quantitative analysis of metastatic tumor weight (E) (*p < 0.05, **p < 0.01, t-test).

Article Snippet: His & Flag-tagged PRMT5 recombinant protein (Sino Biological, Cat: 11074- H18H, China), GST-tagged fusion protein Smad4 (Proteintech, Cat No: Ag0299, USA), and MEP50 recombinant protein (Abnova, H00079084-P01, China) pure proteins were purchased.

Techniques: Methylation, In Vivo, Luciferase, Expressing, Injection, Immunohistochemistry, Mutagenesis

Journal: Blood

Article Title: PRMT5 interacts with the BCL6 oncoprotein and is required for germinal center formation and lymphoma cell survival

doi: 10.1182/blood-2018-02-831438

Figure Lengend Snippet: PRMT5 is required for GC formation and affinity maturation. (A) Immunohistochemistry of paraffin-embedded splenic tissue from Cγ1-Cre−/− Prmt5fl/fl and Cγ1-Cre+/− Prmt5fl/fl mice (n = 7 per group) immunized with SRBCs for 10 days. (B) Quantification of PNA+ clusters from panel A. (C) Representative flow cytometry plot showing percentage of GC B cells (GL7+ FAS+ or CD38dim FAS+) gated on live B220+ splenocytes in Cγ1-Cre−/− Prmt5fl/fl and Cγ1-Cre+/− Prmt5fl/fl mice (n = 7 per group) immunized with SRBC as described in panel A. (D) Titers of low-affinity NP-specific immunoglobulin were measured using NP26-BSA in the serum of Cγ1-Cre−/− Prmt5fl/fl and Cγ1-Cre+/− Prmt5fl/fl mice (n = 6 per group) immunized with NP-CGG for 8 days. (E) Ratio of high- to low-affinity NP-specific immunoglobulin detected with NP4-BSA and NP26-BSA, respectively, in NP-CGG immunized Cγ1-Cre−/− Prmt5fl/fl and Cγ1-Cre+/− Prmt5fl/fl mice described in panel D.

Article Snippet: Recombinant glutathione S -transferase (GST) PRMT5 protein was from Novus Biologicals (Littleton, CO), recombinant BCL6 was from Origene (Rockville, MD), recombinant GST-MEP50 was from Abnova (Walnut, CA), and recombinant human histone H2A and S -adenosyl-methionine (SAM) were from New England Biolabs (Ipswich, MA).

Techniques: Immunohistochemistry, Flow Cytometry

Journal: Blood

Article Title: PRMT5 interacts with the BCL6 oncoprotein and is required for germinal center formation and lymphoma cell survival

doi: 10.1182/blood-2018-02-831438

Figure Lengend Snippet: The arginine methyltransferase PRMT5 directly interacts with BCL6. (A) Co-IP experiments for PRMT5 and BCL6 in 293T cells transfected with BCL6. (B) Co-IP experiments for PRMT5 and BCL6 in SUDHL8 and U2932 DLBCL cell lines. (C) PRMT5 and BCL6 interact in normal CD77+ GC B cells, enriched as described in supplemental Materials and methods. (D) GST pull-down assay of recombinant GST-PRMT5 and BCL6-MYC/DDK proteins. Purified GST-PRMT5 or GST proteins were incubated with BCL6-MYC/DDK protein for 12 hours. The coprecipitated BCL6 and PRMT5 proteins were detected by western blot with anti-GST and anti-BCL6 antibodies.

Article Snippet: Recombinant glutathione S -transferase (GST) PRMT5 protein was from Novus Biologicals (Littleton, CO), recombinant BCL6 was from Origene (Rockville, MD), recombinant GST-MEP50 was from Abnova (Walnut, CA), and recombinant human histone H2A and S -adenosyl-methionine (SAM) were from New England Biolabs (Ipswich, MA).

Techniques: Co-Immunoprecipitation Assay, Transfection, Pull Down Assay, Recombinant, Purification, Incubation, Western Blot

Journal: Blood

Article Title: PRMT5 interacts with the BCL6 oncoprotein and is required for germinal center formation and lymphoma cell survival

doi: 10.1182/blood-2018-02-831438

Figure Lengend Snippet: PRMT5 interacts with the RD2 and ZNF domains of BCL6 via its N terminus. (A) Schematic of plasmids encoding either the V5-tagged full-length BCL6 or domains of BCL6 used in panel B. (B) Co-IP experiments of PRMT5 with V5-tagged full-length or BTB/POZ, RD2 and ZNF domains of BCL6. (C) GST pull-down assay of recombinant GST-PRMT5 with BCL6 RD2-V5/His proteins. (D) V5 pull-down assay of recombinant GST-PRMT5 with BCL6 ZNF-V5 proteins. (E) RD2 domain of BCL6 interacts with the catalytically active N terminus of PRMT5 (residues 1-320).

Article Snippet: Recombinant glutathione S -transferase (GST) PRMT5 protein was from Novus Biologicals (Littleton, CO), recombinant BCL6 was from Origene (Rockville, MD), recombinant GST-MEP50 was from Abnova (Walnut, CA), and recombinant human histone H2A and S -adenosyl-methionine (SAM) were from New England Biolabs (Ipswich, MA).

Techniques: Co-Immunoprecipitation Assay, Pull Down Assay, Recombinant

Journal: Blood

Article Title: PRMT5 interacts with the BCL6 oncoprotein and is required for germinal center formation and lymphoma cell survival

doi: 10.1182/blood-2018-02-831438

Figure Lengend Snippet: PRMT5 and MEP50 mediate the repressive activity of the BCL6 RD2 domain. (A) Schematic of plasmids encoding the GAL4 DNA-binding domain (GAL4DBD) fused to either full-length BCL6 or domains of BCL6 used in panels B and D. (B) GAL4 luciferase reporter assays in 293T cells transfected with plasmids encoding GAL4DBD-BCL6 and PRMT5/MEP50. Western blots demonstrate representative expression of GAL4DBD-BCL6, PRMT5, and MEP50 following expression of corresponding expression vectors. All the experiments were repeated 3 times in triplicate. ***P < .001. (C) 4xBCL6 binding site luciferase reporter assays in 293T cells transfected with plasmids encoding BCL6 and PRMT5/MEP50. Western blots demonstrate representative expression of BCL6, PRMT5, and MEP50 following expression of corresponding expression vectors. All the experiments were repeated 3 times in triplicate. ***P < .001. (D) GAL4 luciferase reporter assays in 293T cells transfected with full-length GAL4DBD-BCL6, GAL4DBD-BTB/POZ, or GAL4DBD-RD2 domains of BCL6 in the presence or absence of cotransfected PRMT5/MEP50 siRNAs from GE Dharmacon (Lafayette, CO). Western blots demonstrate representative expression of PRMT5 and BCL6 or MEP50 and BCL6 following transfection of increasing concentrations of PRMT5 siRNA-1 and MEP50 siRNA-1, respectively. All the experiments were repeated 3 times in triplicate. ***P < .001. Independent siRNA to PRMT5 and MEP50 shown in supplemental Figure 3. (E) 4xBCL6 binding site luciferase reporter assays in 293T cells transfected with plasmids encoding BCL6 alone or in the presence of GSK591 (*P < .05; **P < .01; ***P < .001). (F) 4xBCL6 binding site luciferase reporter assays in 293T cells transfected with plasmids encoding BCL6, PRMT5, or catalytically inactive PRMT5 mutant (G367A/R368A, muPRMT5). Western blots demonstrate representative expression of BCL6, PRMT5, and muPRMT5 (G367A/R368A) following expression of corresponding expression vectors. All the experiments were repeated 3 times in triplicate. *P < .05; **P < .01.

Article Snippet: Recombinant glutathione S -transferase (GST) PRMT5 protein was from Novus Biologicals (Littleton, CO), recombinant BCL6 was from Origene (Rockville, MD), recombinant GST-MEP50 was from Abnova (Walnut, CA), and recombinant human histone H2A and S -adenosyl-methionine (SAM) were from New England Biolabs (Ipswich, MA).

Techniques: Activity Assay, Binding Assay, Luciferase, Transfection, Western Blot, Expressing, Mutagenesis

Journal: Blood

Article Title: PRMT5 interacts with the BCL6 oncoprotein and is required for germinal center formation and lymphoma cell survival

doi: 10.1182/blood-2018-02-831438

Figure Lengend Snippet: PRMT5 directly dimethylates arginines of BCL6 in lymphoma. (A) IP for BCL6 and symmetric arginine dimethylation (SYM10) in lymphoma cell lines OCI-LY1 and Raji reveals symmetric arginine dimethylation of BCL6. (B) Knockdown of PRMT5 with specific siRNA decreases BCL6 symmetric arginine methylation. Raji cells were transfected with PRMT5 or control siRNAs followed by IP with symmetric arginine dimethylation (SYM10) antibody and immunoblotting with BCL6 antibody. Also shown are western blots with indicated antibodies from the same cells. (C) In vitro methyltransferase assay with recombinant PRMT5, MEP50, and BCL6 or H2A proteins. The proteins were blotted with the indicated antibodies. In addition, the reaction mixture was immunoprecipitated with BCL6 antibody and blotted with antibodies for symmetric arginine dimethylation (SYM10) and BCL6. (D) In vitro thymidine incorporation methyltransferase assay with recombinant PRMT5 and BCL6 or H2A proteins.

Article Snippet: Recombinant glutathione S -transferase (GST) PRMT5 protein was from Novus Biologicals (Littleton, CO), recombinant BCL6 was from Origene (Rockville, MD), recombinant GST-MEP50 was from Abnova (Walnut, CA), and recombinant human histone H2A and S -adenosyl-methionine (SAM) were from New England Biolabs (Ipswich, MA).

Techniques: Knockdown, Methylation, Transfection, Control, Western Blot, In Vitro, Recombinant, Immunoprecipitation

Journal: Blood

Article Title: PRMT5 interacts with the BCL6 oncoprotein and is required for germinal center formation and lymphoma cell survival

doi: 10.1182/blood-2018-02-831438

Figure Lengend Snippet: PRMT5 dimethylates BCL6 at R305 to mediate the repressive activity of the BCL6 RD2 domain. (A) Mass spectroscopy of BCL6 protein following in vitro methyltransferase assay by PRMT5 identifies BCL6 methylation at R305. Representative tandem mass spectrometry spectrum of methylated R305 of BCL6 (P11482). Band corresponding to BCL6 was excised from the gel and subjected to trypsin digestion. Tryptic peptides were resolved on a reverse phase column, and high-energy collision dissociation spectra were obtained using Orbitrap Fusion Tribrid mass spectrometer. Data were searched against human protein database using Proteome Discoverer (v 1.4, ThermoScientific) by considering methylation on arginine as a potential modification. Results were filtered at 1% false discovery rate. A tandem mass spectrometry spectrum corresponding to 302EEErPSSEDEIALHFEPPNAPLNR325 of BCL6 (precursor [M+H]+4 = 698.3398 m/z. DPPM = 1.6, inset) is shown. Observed b- and y-ions are indicated. The lowercase “r” is the methylated arginine. (B) 4xBCL6 binding site luciferase reporter assays in 293T cells transfected with wild-type and R305K mutant BCL6 or control pcDNA3.1 vector in the presence or absence of cotransfected siRNAs to PRMT5. ***P < .001. Western blots in each experimental condition with the indicated antibodies. (C) Arginine dimethylation of wild-type or R305K BCL6 transfected into Raji cells. (D) Co-IP experiments of endogenous PRMT5 with wild-type or R305K BCL6 transfected into Raji and 293T cells. WT, wild-type.

Article Snippet: Recombinant glutathione S -transferase (GST) PRMT5 protein was from Novus Biologicals (Littleton, CO), recombinant BCL6 was from Origene (Rockville, MD), recombinant GST-MEP50 was from Abnova (Walnut, CA), and recombinant human histone H2A and S -adenosyl-methionine (SAM) were from New England Biolabs (Ipswich, MA).

Techniques: Activity Assay, Mass Spectrometry, In Vitro, Methylation, Modification, Binding Assay, Luciferase, Transfection, Mutagenesis, Control, Plasmid Preparation, Western Blot, Co-Immunoprecipitation Assay

Journal: Blood

Article Title: PRMT5 interacts with the BCL6 oncoprotein and is required for germinal center formation and lymphoma cell survival

doi: 10.1182/blood-2018-02-831438

Figure Lengend Snippet: PRMT5 recruits BCL6 to its target genes to induce gene repression. (A) Enrichment of BCL6, SMRT, HA-tagged PRMT5, and IgG at BCL6 targets in OCI-LY1 cells treated with vehicle or 200 nM GSK591 for 72 hours. *P < .05; **P < .01. (B) Messenger RNA abundance of BCL6 target genes in OCI-LY1, OCI-LY7, SUDHL4, and SUDHL6 cells treated with vehicle or 200 nM GSK591 for 96 hours. *P < .05; **P < .01. (C) Immunoblot demonstrating inhibition of SDMA using the SYM10 antibody at 72 hours after treatment with 200 nM GSK591. IP demonstrating decrease in symmetric dimethylation of BCL6 shown in supplemental Figure 7A. (D) Pathway analysis of gene expression changes in SUDHL6 cells treated with vehicle or 200 nM GSK591 for 24 or 96 hours. The Fisher exact test was used to calculate enrichment P values for each gene set, and the Benjamini-Hochberg method was used for false discovery rate control. (E) GSK591 concentration that results in 50% growth inhibition (GI50) of BCL6-dependent and BCL6-independent DLBCL cell lines treated with vehicle or increasing concentrations of GSK591 for 6 days. Raw growth inhibition curves of GSK591 alone are shown in supplemental Figure 8A. (F) Messenger RNA abundance of BCL6 targets in OCI-LY1 cells with combined treatment of 25 μM FX1 and 200 nM GSK591 for 48 hours. *P < .05; **P < .01 relative to vehicle. †P < 0.05; ††P < .01 relative to each drug alone. (G) Combination indexes of the BCL6 inhibitor FX1 with GSK591 after treating cells with increasing concentrations of GSK591 for 6 days and FX1 for 2 days. Data are representative of 3 triplicates ± standard error of the mean (SEM). Raw growth inhibition curves of each drug alone and in combination are shown in supplemental Figure 8C. (H) Mean fluorescence intensity of carboxyfluorescein diacetate succinimidyl ester of live CD19+ (GhostDye− or DAPI−) human DLBCL samples on day 6 after cells were exposed to GSK591 on day 0 then treated with FX1 3 days later. Data representative of 3 triplicates ± SEM. *P < .05; **P < .01 relative to VEH. †P < 0.05; ††P < .01 relative to each drug alone. (I) Cell viability (GhostDye− or DAPI−) of CD19+ human DLBCL from panel H. Data are representative of 3 triplicates ± SEM. *P < .05; **P < .01 relative to vehicle. †P < .05; ††P < .01 relative to each drug alone. FACS, fluorescence-activated cell sorting; FL, follicular lymphoma; MFI, mean fluorescence intensity; MHC, major histocompatibility complex; mRNA, messenger RNA; VEH, vehicle.

Article Snippet: Recombinant glutathione S -transferase (GST) PRMT5 protein was from Novus Biologicals (Littleton, CO), recombinant BCL6 was from Origene (Rockville, MD), recombinant GST-MEP50 was from Abnova (Walnut, CA), and recombinant human histone H2A and S -adenosyl-methionine (SAM) were from New England Biolabs (Ipswich, MA).

Techniques: Western Blot, Inhibition, Gene Expression, Control, Concentration Assay, Fluorescence, FACS, Immunopeptidomics

Journal: Science signaling

Article Title: Protein Arginine Methyltransferase 5 Regulates ERK1/2 Signal Transduction Amplitude and Cell Fate Through CRAF

doi: 10.1126/scisignal.2001936

Figure Lengend Snippet: PRMT5 depletion increases ERK1/2 phosphorylation in response to growth factors. (A) Knockdown of Prmt5 in 37–31E, PC12, or MEFs enhances the increase in ERK1/2 phosphorylation produced by growth factor treatment. Cells transfected with either scrambled siRNA (Scr.) or Prmt5 siRNA were serum-starved and exposed to MTA for 3 hours and then treated with HGF for 10 min. p-ERK1/2, ERK1/2, and PRMT5 abundance was assessed by Western blot. (B) Overexpression of the catalytically inactive PRMT5 mutant (PRMT5ΔGAGRG) in HEK293 cells reproduces the effects of MTA on ERK1/2 phosphorylation. Fortyeight hours after transfection with either empty vector or pEF2-PRMT5ΔGAGRG expression vector, cells were serumstarved and pretreated with MTA for 2 hours and then stimulated with HGF for 10 min. Cell lysates were assessed for p-ERK1/2, ERK1/2, PRMT5, and GAPDH by Western blotting. (C) Growth factors regulate PRMT5 methyltransferase activity. Cos-7 cells stably transfected with Flag-PRMT5 were serum-starved for 3 hours and then treated with the indicated growth factors for 10 min. After PRMT5 immunoprecipitation, its methyltransferase activity was measured in vitro. The3H radioactive signal incorporated in MBP was assessed autoradiographically after SDS-PAGE. Immunoprecipitated PRMT5 and MBP are shown as loading controls. Graph shows quantification of methyltransferase activity normalized by the amount of PRMT5 immunoprecipitated. P value was calculated with Student’s t test (n = 3 independent replicates).

Article Snippet: Recombinant PRMT5 (2 μg; Novus Biologicals) and recombinant full-length inactive GST-CRAF (2 μg; Sigma-Aldrich) or active GST-CRAF (N-terminal GST-tagged, residues 306 to end) (2 μg; GeneScript Inc.) were incubated for 1 hour at 30°C in a final volume of 50 μl of methylation buffer [100 mM tris-HCl (pH8.0), 1 mM EDTA, and 1 mMDTT].

Techniques: Phospho-proteomics, Knockdown, Produced, Transfection, Western Blot, Over Expression, Mutagenesis, Plasmid Preparation, Expressing, Activity Assay, Stable Transfection, Immunoprecipitation, In Vitro, SDS Page

Journal: Science signaling

Article Title: Protein Arginine Methyltransferase 5 Regulates ERK1/2 Signal Transduction Amplitude and Cell Fate Through CRAF

doi: 10.1126/scisignal.2001936

Figure Lengend Snippet: MTA and PRMT5 depletion increases CRAF’s catalytic activity. (A) 37–31E cells were serum-starved, pretreated with MTA for 3 hours, and then treated with HGF for 5 min. c-Met was immunoprecipitated from total lysates. Western blot shows the amount of p-c-Met in the immunocomplexes. p-ERK1/2 and ERK1/2 levels in the samples used in the immunoprecipitation are shown (Input). (B) RAS activation is not affected by MTA. 37–31E cells were treated as in (A). RAS-GTP was isolated by affinity chromatography with GST-RBD (GST–RAS binding domain of CRAF). Complexes were separated by SDS-PAGE. Western blot shows the amount of RAS-GTP. p-ERK1/2 and ERK1/2 demonstrate the activation of the pathway in the samples used. (C) 37–31E cells transfected with CRaf siRNA were serumstarved and pretreated with MTA for 2 hours and then triggered with HGF for 10 min. Western blot shows CRAF and p-ERK1/2. P value was calculated with Student’s t test (n = 3 different experiments). (D) PRMT5 was immunoprecipitated from total lysates of HEK293 cells transfected with either scrambled (Scr.) or PRMT5 siRNA. Western blots show PRMT5 and CRAF in the immunocomplexes and PRMT5 in the initial total lysates (left panel). Endogenous PRMT5 and CRAF were immunoprecipitated from lysates of Cos-7 cells untreated or treated with EGF. Western blots assess the presence of PRMT5 in the immunocomplexes. (E) PRMT5 binds to active CRAF. Recombinant PRMT5 was incubated with either full-length inactive GST-CRAF or active GST-RAF (N-terminal GST-tagged, residues 306 to end). Western blot shows CRAF after PRMT5 immunoprecipitation. Immunoprecipitated PRMT5 is shown as loading control. (F) MTA increases growth factor–induced CRAF catalytic activity. HeLa cells transiently transfected with Flag-CRAFWT were serum-starved, treated with MTA for 3 hours, and then treated with either EGF or HGF for 10 min. Immunoprecipitated Flag-CRAF was used to perform an in vitro kinase assay. Graph shows CRAF catalytic activity (fold induction over untreated cells) in a coupled RAF-MEK-ERK-MBP radioactive assay under the different conditions (left). Western blots were performed against p-MEK under the same conditions (right). P value was calculated with Student’s t test (n = 3 replicates). (G) MTA increases the endogenous CRAF catalytic activity in response to growth factors. MEFs were starved and treated with MTA for 3 hours, and cells were exposed to EGF for 10 min. Immunoprecipitated endogenous CRAF was used to perform an in vitro kinase assay as in (F). Graphs show the CRAF catalytic activity fold induction over untreated cells. A radioactive assay and Western blots show the p-MEK levels induced by immunoprecipitated CRAF. Bars indicate the SD. P value was calculated with Student’s t test (n = 3 replicates). (H) 37–31E cells transfected with either scrambled (Scr.) or Prmt5 siRNA were treated as in (F). Graph shows the kinase activity of endogenous CRAF. Western blots show the p-MEK levels induced by immunoprecipitated CRAF. PRMT5 protein levels in total lysates used in the immunoprecipitation are shown. Bars indicate the SD. P value was calculated with Student’s t test (n = 3 replicates).

Article Snippet: Recombinant PRMT5 (2 μg; Novus Biologicals) and recombinant full-length inactive GST-CRAF (2 μg; Sigma-Aldrich) or active GST-CRAF (N-terminal GST-tagged, residues 306 to end) (2 μg; GeneScript Inc.) were incubated for 1 hour at 30°C in a final volume of 50 μl of methylation buffer [100 mM tris-HCl (pH8.0), 1 mM EDTA, and 1 mMDTT].

Techniques: Activity Assay, Immunoprecipitation, Western Blot, Activation Assay, Isolation, Affinity Chromatography, Binding Assay, SDS Page, Transfection, Recombinant, Incubation, Control, In Vitro, Kinase Assay, Radioactivity

Journal: Science signaling

Article Title: Protein Arginine Methyltransferase 5 Regulates ERK1/2 Signal Transduction Amplitude and Cell Fate Through CRAF

doi: 10.1126/scisignal.2001936

Figure Lengend Snippet: Arginine-to-lysine mutation in the GRG motif of RAF proteins promotes their stability and amplifies their increase in kinase activity in response to growth factors. (A) In vivo methylation assay in PC12 cells. Cells transiently transfected with either scrambled or Prmt5 siRNA and metabolically labeled ([3H]methionine) were serum-starved and pretreated with MTA, and then cells were exposed to EGF for 10 min. After immunoprecipitation of endogenous CRAF, the3H radioactive signal incorporated in CRAF was assessed autoradiographically after SDS-PAGE. The same membranes were assessed for CRAF (loading control). PRMT5 in total lysates is shown in the lower panel. (B) Sequence alignment of RAF isoforms from different species showing the conserved GRG motif (left). A 3D image made with UCSF Chimera (http://www.cgl.ucsf.edu/chimera) and the 1UWH file from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (http://www.pdb.org) show the location of the Arg671 residue in the BRAF 3D structure (right). (C) HEK293 cells were transiently transfected with Flag-CRAFWT or Flag-CRAFR563K as indicated. Western blot shows p-ERK1/2 and abundance of transfected proteins. Quantification of p-ERK1/2 normalized by Ponceau S staining is shown (fold induction with respect to Flag-CRAFWT–transfected untreated cells). (D) Catalytic activity of CRAFR563K mutant compared to that of the wild-type isoform. HeLa cells were transfected with Flag-CRAFWT or one-sixth of the amount of the Flag-CRAFR563K mutant. CRAF catalytic activity was assessed in immunoprecipitates. Graph shows the CRAF catalytic activity (fold induction over untreated cells) in a coupled RAF-MEK-ERK-MBP radioactive assay under the different conditions (top). P value was calculated with Student’s t test (n = 3 replicates). Western blots show p-MEK under the same conditions (lower panel). (E) SKMel147 cells were transiently transfected with scrambled siRNA (Scr.) or PRMT5 siRNA (siPRMT5) for 24 hours. Cells were pulse-labeled with [35S]Met and re-collected over a 24-hour time course (upper left panels). Cell lysates were harvested and immunoprecipitated with antibody directed against CRAF; after SDS-PAGE separation and capture of the35S signal, CRAF was quantitated by autoradiography. Left, data presented as autoradiogram; right, data presented graphically. Cells were treated with CHX (5 μg/ml) and collected at the indicated time points (lower panel). Immunoprecipitation of CRAF was performed from 500 μg of total protein. Western blots show CRAF and p-CRAFS621. Graph shows CRAF quantifications. Dashed line represents 100%. Amounts of PRMT5 are shown in the input samples.

Article Snippet: Recombinant PRMT5 (2 μg; Novus Biologicals) and recombinant full-length inactive GST-CRAF (2 μg; Sigma-Aldrich) or active GST-CRAF (N-terminal GST-tagged, residues 306 to end) (2 μg; GeneScript Inc.) were incubated for 1 hour at 30°C in a final volume of 50 μl of methylation buffer [100 mM tris-HCl (pH8.0), 1 mM EDTA, and 1 mMDTT].

Techniques: Mutagenesis, Activity Assay, In Vivo, Methylation, Transfection, Metabolic Labelling, Labeling, Immunoprecipitation, SDS Page, Control, Sequencing, Residue, Western Blot, Staining, Radioactivity, Autoradiography

Journal: Science signaling

Article Title: Protein Arginine Methyltransferase 5 Regulates ERK1/2 Signal Transduction Amplitude and Cell Fate Through CRAF

doi: 10.1126/scisignal.2001936

Figure Lengend Snippet: PRMT5 methylates CRAF on Arg563. (A) HeLa cells were transfected with either Flag-CRAFWT or Flag-CRAFR563K for 48 hours. Cells were starved and treated with MTA for 2 hours and then treated with EGF for 10 min. Subsequently, Flag-tagged CRAF proteins were immunoprecipitated and the complexes were eluted with M2 peptide. A second immunoprecipitation was performed on the eluates with the anti-sDMA antibody SYM10. The immunocomplexes were analyzed for the presence of CRAF by Western blot and mass spectrometry (MS). Two different antibodies against CRAF were used. Coomassie blue staining of the gel showing the band corresponding to the size of CRAF (*) analyzed by liquid chromatography–mass spectrometry is shown. Table shows the sequences of the identified peptides corresponding to the indicated proteins (lower panel). (B) PRMT5 depletion inhibits CRAF methylation. HeLa cells transfected with either scrambled or PRMT5 siRNA for 48 hours and Flag-CRAFWT for an extra 24 hours were starved for 2 hours and treated with EGF. Lysates were processed according to the protocol described in (A). Western blot shows the abundance of CRAF. Right panel shows PRMT5 in the initial total lysates. (C) CRAFR563K is not methylated after treatment with growth factors. HeLa or HEK293 cells transfected with either Flag-CRAFWT or Flag-CRAFR563K were metabolically labeled with [3H]methionine and treated with EGF for 10 min. After immunoprecipitation with the α-Flag antibody, the3H radioactive signal incorporated in CRAF was assessed autoradiographically after SDS-PAGE. Immunoprecipitated CRAF is shown as loading control. Graph shows quantification of CRAF isoform methylation relative to untreated cells. P value was calculated with Student’s t test (n = 3 replicates). (D) CRAF peptide containing GRG motif is methylated in vitro. In vitro methylation assay with immunoprecipitated PRMT5 from EGF-treated Cos-7 cells stably transfected with PRMT5. Assay was performed adding either no peptide, FMVGRGYAS peptide, or FMVGKGYAS mutant peptide as a control for specificity of arginine methylation. The fragmentation spectrum of the methylated peptide identified by MALDI-TOF/TOF mass spectrometry is shown in the bottom panel. m/z, mass/charge ratio.

Article Snippet: Recombinant PRMT5 (2 μg; Novus Biologicals) and recombinant full-length inactive GST-CRAF (2 μg; Sigma-Aldrich) or active GST-CRAF (N-terminal GST-tagged, residues 306 to end) (2 μg; GeneScript Inc.) were incubated for 1 hour at 30°C in a final volume of 50 μl of methylation buffer [100 mM tris-HCl (pH8.0), 1 mM EDTA, and 1 mMDTT].

Techniques: Transfection, Immunoprecipitation, Western Blot, Mass Spectrometry, Staining, Liquid Chromatography, Methylation, Metabolic Labelling, Labeling, SDS Page, Control, In Vitro, Stable Transfection, Mutagenesis

Journal: Science signaling

Article Title: Protein Arginine Methyltransferase 5 Regulates ERK1/2 Signal Transduction Amplitude and Cell Fate Through CRAF

doi: 10.1126/scisignal.2001936

Figure Lengend Snippet: Model for the limitation of the ERK1/2 signal by PRMT5. Growth factors or mutated RAS activate CRAF and thereby the MAPK signaling pathway. PRMT5 promotes CRAF degradation and limits its catalytic activity, reducing the activation of downstream kinases, such as MEK1/2 and ERK1/2. In contrast, PRMT5 does not affect downstream signals in cells with BRAFV600E, which are independent of RAS. Decreasing PRMT5 activity (pharmacologically or by its knockdown) increases the stability of activated CRAF, increasing the amplitude of the ERK1/2 signal and the integrated signal strength and consequently affecting the evoked biological response.

Article Snippet: Recombinant PRMT5 (2 μg; Novus Biologicals) and recombinant full-length inactive GST-CRAF (2 μg; Sigma-Aldrich) or active GST-CRAF (N-terminal GST-tagged, residues 306 to end) (2 μg; GeneScript Inc.) were incubated for 1 hour at 30°C in a final volume of 50 μl of methylation buffer [100 mM tris-HCl (pH8.0), 1 mM EDTA, and 1 mMDTT].

Techniques: Activity Assay, Activation Assay, Knockdown

Journal: Journal of Biological Chemistry

Article Title: CARM1-mediated methylation of protein arginine methyltransferase 5 represses human γ-globin gene expression in erythroleukemia cells

doi: 10.1074/jbc.ra118.004028

Figure Lengend Snippet: Figure 2. Methylation of PRMT5 at R505 is essential for its methyltransferase activity. (A) Western blot analysis of extracts from K562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K or PRMT5Δ using FLAG and PRMT5 antibodies. GAPDH was used as a loading control. Blots are representative of three independent experiments. (B) Quantitative real-time PCR analysis of PRMT5 mRNA normalized to β-actin in K562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K or PRMT5Δ. (C) Western blot analysis of extracted histones from K562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K or PRMT5Δ using H4R3me2s antibody. Histone H4 was used as a loading control. Blots are representative of three independent experiments. (D) ChIP analysis of H4R3me2s enrichment at the γ-promoter in K562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K or PRMT5Δ. The results are shown as the means ± SD from three independent experiments. Two-tailed Student’s t-tests were used to compare means. *P < 0.05, **P < 0.01 compared to the vector control. (E) Quantitative real-time PCR analysis of -globin mRNA normalized to β-actin in K562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K or PRMT5Δ. The results are shown as the means ± SD from three independent experiments. Two-tailed Student’s t-tests were used to compare means. **P < 0.01 compared to the vector control.

Article Snippet: Briefly, we incubated these enzymes with 5 g of purified recombinant PRMT5 and 2 mCi of the methyl donor, S-adenosyl-L-methyl-3H-methionine (3H-SAM, PerkinElmer), in 20 l of HMTase buffer (25 mM NaCl, 25 mM Tris-HCl, pH 8.8) for 2 hours at 30°C.

Techniques: Methylation, Activity Assay, Western Blot, Plasmid Preparation, Control, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Journal of Biological Chemistry

Article Title: CARM1-mediated methylation of protein arginine methyltransferase 5 represses human γ-globin gene expression in erythroleukemia cells

doi: 10.1074/jbc.ra118.004028

Figure Lengend Snippet: Figure 7. Arg505 methylation is essential for PRMT5 oligomerization. Western blot analysis using PRMT5 antibody to detect PRMT5 oligomerization in K562 cells overexpressing PRMT5-WT, PRMT5-R505A or PRMT5-R505K treated with PBS control (A) or the crosslinker EDC+NHS (B). The asterisk and the hash indicate the PRMT5 dimer and PRMT5 tetramer, respectively.

Article Snippet: Briefly, we incubated these enzymes with 5 g of purified recombinant PRMT5 and 2 mCi of the methyl donor, S-adenosyl-L-methyl-3H-methionine (3H-SAM, PerkinElmer), in 20 l of HMTase buffer (25 mM NaCl, 25 mM Tris-HCl, pH 8.8) for 2 hours at 30°C.

Techniques: Methylation, Western Blot, Control