recombinant prmt1  (Revvity)

Journal: Cell reports

Article Title: Opposing roles of p38α-mediated phosphorylation and PRMT1-mediated arginine methylation in driving TDP-43 proteinopathy.

doi: 10.1016/j.celrep.2024.115205

Figure Lengend Snippet: Figure 4. TDP-43 is methylated by PRMT1 at R293 (A) Sequence alignment of amino acids 287–307 of TDP-43 from diverse vertebrates. Conserved 292– 293 sites are bolded. S292 phosphorylation site and R293-G295 RGG-motif are highlighted in yellow and green, respectively. (B) Western blot of immunoprecipitated endoge- nous TDP-43 from SH-SY5Y-cells probed with antibodies against TDP-43, ADMA, MMA, and SDMA. TDP-43 with arginine methylation is marked by arrowheads. (C) Western blot of immunoprecipitated endoge- nous TDP-43 from SH-SY5Y-cells with siRNA- induced PRMT1 knockdown probed with anti- bodies against TDP-43 or MMA. A PRMT1 blot was run separately to confirm siRNA-mediated knockdown of PRMT1. (D) Western blot of expressed and immunopre- cipitated TDP-43WT using anti-FLAG antibody from SH-SY5Y-cells treated with DMSO (D) or with methyltransferase inhibitor AdOx (A) at a final concentration of 20 mM for 24 h. FL, full-length TDP-43; CTF35, C-terminal 35-kDa fragment of TDP-43. (E) Western blot of expressed and immunopre- cipitated TDP-43WT and TDP-43R293K using anti- FLAG antibody from SH-SY5Y cells probed with antibodies against TDP-43 and MMA. See also Figures S6 and S7.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Recombinant human PRMT1 protein Abcam ab89007 S-(50-Adenosyl)-L-methionine chloride dihydrochloride Sigma A7007 mTESR media StemCell Technologies Cat # 05825 ROCK inhibitor Selleck Chemicals Cat #S1049 Growth factor-reduced matrigel Corning Cat # 356231 Versene Gibco Cat # 5040066 DBPS Thermo Fisher Cat # 21-031-CV Accutase Thermo Fisher Cat # NC9839010 Poly-L-ornithine solution Sigma Cat # RNBK5888 Mouse laminin Sigma Cat #L2020 FGF R@D Cat # 233-FB Stem-Cell Banker GMP Grade CedarLane Labs Cat # 11890 Fibronectin Corning Cat # CB40008A Neurobasal media Gibco Cat # 12348017 NEAA Gibco Cat # 11-140-050 Glutamax Gibco Cat # 35050061 N2 Gibco Cat # 17502048 B27 Gibco Cat # 17504044 SB431542 StemCell Technologies Cat # 72234 LDN193189 Sigma-Aldrich Cat # SML0559 Retinoic Acid Sigma-Aldrich Cat #R2625 Smoothened-Agonist (SAG) Cayman Chemical Cat # 11914 DAPT Cayman Chemical Cat # 13197 BDNF PeproTech Cat # 450-02 GDNF PeproTech Cat # 450-10 CNTF PeproTech Cat # 450-13 Ascorbic acid Sigma-Aldrich Cat # A4403 DMSO Sigma-Aldrich Cat #D4540 OptiMEM Gibco Cat # 31985070 PBS Gibco Cat # 10010023 Paraformaldehyde Electron Microscopy Sciences Cat # 15714-S Donkey Serum Jackson ImmunoResearch Cat # 017-000-121 Prolong Glass mounting media Invitrogen Cat #P36981 Trypan Blue Solution, 0.4% Gibco Cat # 15250061 cOmpleteTM, Mini, EDTA-free Protease Inhibitor Cocktail Millipore Sigma Cat # 4693159001 NuPAGE MES SDS Running Buffer (20X) ThermoFisher Scientific Cat # NP0002 NuPAGE Bis-Tris Mini Protein Gels, 4–12%, 1.5 mm ThermoFisher Scientific Cat # NP0335BOX PierceTM Reversible Protein Stain Kit for Nitrocellulose Membranes Thermo Scientific Cat # PI24580 Trans-Blot Turbo Mini 0.2 mm Nitrocellulose Transfer Packs Bio-Rad Cat # 1704158 SuperSignalTM West Pico PLUS Chemiluminescent Substrate Thermo Scientific Cat # PI34577 Critical commercial assays Pierce BCA Protein Assay Kit ThermoFisher Scientific Cat # 23225 Z’-Lyte kinase assay for p38a ThermoFisher Scientific Cat # PV3177 Pierce LDH Cytotoxicity Assay Kit ThermoFisher Scientific Cat # 88953 (Continued on next page) Cell Reports 44, 115205, January 28, 2025 23

Techniques: Methylation, Sequencing, Phospho-proteomics, Western Blot, Immunoprecipitation, Knockdown, Concentration Assay

Journal: Cell reports

Article Title: Opposing roles of p38α-mediated phosphorylation and PRMT1-mediated arginine methylation in driving TDP-43 proteinopathy.

doi: 10.1016/j.celrep.2024.115205

Figure Lengend Snippet: Figure 6. Arginine methylation of TDP-43 regulates its aggregation (A) Western blot of total and pTDP-43 in RIPA and urea fractions of TDP-43WT-transfected SH-SY5Y-cells treated with DMSO (D) or with methyltransferase inhibitor AdOx (A) at a final concentration of 20 mM for 24 h. The positions of FLAG-tagged TDP-43 (exo) and endogenous TDP-43 (endo) are indicated. (B) Quantification of urea/RIPA ratio of total TDP-43 normalized to levels in DMSO-treated cells (mean ± SD, unpaired t test, n = 4). *p < 0.05. (C) Quantification of pTDP-43 in the urea fraction normalized to levels in DMSO-treated cells (mean ± SD, unpaired t test, n = 4). (D) Western blot of total and pTDP-43 in RIPA and urea fractions of TDP-43WT-transfected SH-SY5Y-cells co-transfected with empty control plasmid (Ctrl) or PRMT1. The positions of FLAG-tagged TDP-43 (exo) and endogenous TDP-43 (endo) are indicated. The positions of Myc-tagged PRMT1 (exo) and endogenous PRMT1 (endo) are also indicated. (E) Quantification of urea/RIPA ratio of total TDP-43 normalized to levels in Ctrl-plasmid co-transfected cells (mean ± SD, unpaired t test, n = 5). ***p < 0.001. (F) Quantification of pTDP-43 in the urea fraction normalized to levels in control cells (mean ± SD, unpaired t test, n = 5). ****p < 0.0001. See also Figure S7.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Recombinant human PRMT1 protein Abcam ab89007 S-(50-Adenosyl)-L-methionine chloride dihydrochloride Sigma A7007 mTESR media StemCell Technologies Cat # 05825 ROCK inhibitor Selleck Chemicals Cat #S1049 Growth factor-reduced matrigel Corning Cat # 356231 Versene Gibco Cat # 5040066 DBPS Thermo Fisher Cat # 21-031-CV Accutase Thermo Fisher Cat # NC9839010 Poly-L-ornithine solution Sigma Cat # RNBK5888 Mouse laminin Sigma Cat #L2020 FGF R@D Cat # 233-FB Stem-Cell Banker GMP Grade CedarLane Labs Cat # 11890 Fibronectin Corning Cat # CB40008A Neurobasal media Gibco Cat # 12348017 NEAA Gibco Cat # 11-140-050 Glutamax Gibco Cat # 35050061 N2 Gibco Cat # 17502048 B27 Gibco Cat # 17504044 SB431542 StemCell Technologies Cat # 72234 LDN193189 Sigma-Aldrich Cat # SML0559 Retinoic Acid Sigma-Aldrich Cat #R2625 Smoothened-Agonist (SAG) Cayman Chemical Cat # 11914 DAPT Cayman Chemical Cat # 13197 BDNF PeproTech Cat # 450-02 GDNF PeproTech Cat # 450-10 CNTF PeproTech Cat # 450-13 Ascorbic acid Sigma-Aldrich Cat # A4403 DMSO Sigma-Aldrich Cat #D4540 OptiMEM Gibco Cat # 31985070 PBS Gibco Cat # 10010023 Paraformaldehyde Electron Microscopy Sciences Cat # 15714-S Donkey Serum Jackson ImmunoResearch Cat # 017-000-121 Prolong Glass mounting media Invitrogen Cat #P36981 Trypan Blue Solution, 0.4% Gibco Cat # 15250061 cOmpleteTM, Mini, EDTA-free Protease Inhibitor Cocktail Millipore Sigma Cat # 4693159001 NuPAGE MES SDS Running Buffer (20X) ThermoFisher Scientific Cat # NP0002 NuPAGE Bis-Tris Mini Protein Gels, 4–12%, 1.5 mm ThermoFisher Scientific Cat # NP0335BOX PierceTM Reversible Protein Stain Kit for Nitrocellulose Membranes Thermo Scientific Cat # PI24580 Trans-Blot Turbo Mini 0.2 mm Nitrocellulose Transfer Packs Bio-Rad Cat # 1704158 SuperSignalTM West Pico PLUS Chemiluminescent Substrate Thermo Scientific Cat # PI34577 Critical commercial assays Pierce BCA Protein Assay Kit ThermoFisher Scientific Cat # 23225 Z’-Lyte kinase assay for p38a ThermoFisher Scientific Cat # PV3177 Pierce LDH Cytotoxicity Assay Kit ThermoFisher Scientific Cat # 88953 (Continued on next page) Cell Reports 44, 115205, January 28, 2025 23

Techniques: Methylation, Western Blot, Transfection, Concentration Assay, Control, Plasmid Preparation

Journal: Cell reports

Article Title: Opposing roles of p38α-mediated phosphorylation and PRMT1-mediated arginine methylation in driving TDP-43 proteinopathy.

doi: 10.1016/j.celrep.2024.115205

Figure Lengend Snippet: Figure 7. Crosstalk between PRMT1-catalyzed arginine methylation and p38a-mediated phosphorylation of TDP-43 (A) Western blot of immunoprecipitated FLAG-tagged TDP-43 from SH-SY5Y cells probed with antibodies against TDP-43, pTDP-43, and MMA. GAPDH serves as a loading control. The positions of FLAG-tagged TDP-43 (exo) and endogenous TDP-43 (endo) are indicated. (B) Western blot of immunoprecipitated FLAG-tagged TDP-43WT from SH-SY5Y cells with or without siRNA-induced p38a knockdown probed with antibodies against TDP-43, p38a, pTDP-43, and MMA. GAPDH serves as a loading control. (C) Quantification of TDP-43-CTF/full length-ratio (mean ± SD, unpaired t test, n = 3). **p < 0.01. (D) Quantification of mono-methylated TDP-43-CTF normalized to total TDP-43-CTF (mean ± SD, unpaired t test, n = 3). *p < 0.05. (E) Western blot of immunoprecipitated FLAG-tagged TDP-43WT from SH-SY5Y cells with or without PRMT1 overexpression probed with antibodies against TDP- 43, pTDP-43, and MMA. GAPDH serves as a loading control. The positions of FLAG-tagged TDP-43 (exo) and endogenous TDP-43 (endo) are indicated. The positions of Myc-tagged PRMT1 (exo) and endogenous PRMT1 (endo) are also indicated. See also Figure S7.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Recombinant human PRMT1 protein Abcam ab89007 S-(50-Adenosyl)-L-methionine chloride dihydrochloride Sigma A7007 mTESR media StemCell Technologies Cat # 05825 ROCK inhibitor Selleck Chemicals Cat #S1049 Growth factor-reduced matrigel Corning Cat # 356231 Versene Gibco Cat # 5040066 DBPS Thermo Fisher Cat # 21-031-CV Accutase Thermo Fisher Cat # NC9839010 Poly-L-ornithine solution Sigma Cat # RNBK5888 Mouse laminin Sigma Cat #L2020 FGF R@D Cat # 233-FB Stem-Cell Banker GMP Grade CedarLane Labs Cat # 11890 Fibronectin Corning Cat # CB40008A Neurobasal media Gibco Cat # 12348017 NEAA Gibco Cat # 11-140-050 Glutamax Gibco Cat # 35050061 N2 Gibco Cat # 17502048 B27 Gibco Cat # 17504044 SB431542 StemCell Technologies Cat # 72234 LDN193189 Sigma-Aldrich Cat # SML0559 Retinoic Acid Sigma-Aldrich Cat #R2625 Smoothened-Agonist (SAG) Cayman Chemical Cat # 11914 DAPT Cayman Chemical Cat # 13197 BDNF PeproTech Cat # 450-02 GDNF PeproTech Cat # 450-10 CNTF PeproTech Cat # 450-13 Ascorbic acid Sigma-Aldrich Cat # A4403 DMSO Sigma-Aldrich Cat #D4540 OptiMEM Gibco Cat # 31985070 PBS Gibco Cat # 10010023 Paraformaldehyde Electron Microscopy Sciences Cat # 15714-S Donkey Serum Jackson ImmunoResearch Cat # 017-000-121 Prolong Glass mounting media Invitrogen Cat #P36981 Trypan Blue Solution, 0.4% Gibco Cat # 15250061 cOmpleteTM, Mini, EDTA-free Protease Inhibitor Cocktail Millipore Sigma Cat # 4693159001 NuPAGE MES SDS Running Buffer (20X) ThermoFisher Scientific Cat # NP0002 NuPAGE Bis-Tris Mini Protein Gels, 4–12%, 1.5 mm ThermoFisher Scientific Cat # NP0335BOX PierceTM Reversible Protein Stain Kit for Nitrocellulose Membranes Thermo Scientific Cat # PI24580 Trans-Blot Turbo Mini 0.2 mm Nitrocellulose Transfer Packs Bio-Rad Cat # 1704158 SuperSignalTM West Pico PLUS Chemiluminescent Substrate Thermo Scientific Cat # PI34577 Critical commercial assays Pierce BCA Protein Assay Kit ThermoFisher Scientific Cat # 23225 Z’-Lyte kinase assay for p38a ThermoFisher Scientific Cat # PV3177 Pierce LDH Cytotoxicity Assay Kit ThermoFisher Scientific Cat # 88953 (Continued on next page) Cell Reports 44, 115205, January 28, 2025 23

Techniques: Methylation, Phospho-proteomics, Western Blot, Immunoprecipitation, Control, Knockdown, Over Expression