recombinant prmt1  (BPS Bioscience)

Journal: Nature Communications

Article Title: FBXO7 ubiquitinates PRMT1 to suppress serine synthesis and tumor growth in hepatocellular carcinoma

doi: 10.1038/s41467-024-49087-2

Figure Lengend Snippet: a Schematic of the screening workflow to identify the E3 ubiquitin ligase FBXO7 as a protein candidate downregulating PRMT1. b Venn diagram showing overlap of proteins among four datasets obtained from BioGRID, GSEA-MSIGDB, MGI, and TCGA. c , d Reciprocal co-IP analysis of PRMT1 and FBXO7 in Huh7 and PLC/PRF/5 cells. IgG was used as a negative control. The immunoblotting experiments were repeated three times with similar results. e , f Reciprocal co-IP analysis of PRMT1 and FBXO7 in PRMT1 KD cells. The immunoblotting experiments were repeated three times with similar results. g Recombinant GST-FBXO7 protein was incubated with recombinant His-MBP-PRMT1 protein, followed by GST pulldown and immunoblotting analysis with GST and His antibodies. The immunoblotting experiments were repeated three times with similar results. h Schematic representation of full-length (FL) PRMT1 and different truncation mutants. i , j GFP-PRMT1 FL or indicated truncation mutants were co-expressed with FLAG-FBXO7 in HEK293T cells. FLAG-FBXO7 was immunoprecipitated with FLAG beads, followed by immunoblotting analysis of GFP-PRMT1 using GFP antibody. The immunoblotting experiments were repeated three times with similar results. k Schematic representation of full-length (FL) FBXO7 and different truncation mutants. UBL ubiquitin-like domain, FP FBXO7-PI31 dimerization domain, PRR proline-rich region. l , m FLAG-FBXO7 FL or indicated truncation mutants were co-expressed with GFP-PRMT1 in HEK293T cells. FLAG-FBXO7 was immunoprecipitated with FLAG beads, followed by immunoblotting analysis of GFP-PRMT1 using GFP antibody. The immunoblotting experiments were repeated three times with similar results. Source data are provided as a Source Data file.

Article Snippet: Recombinant human GST-FBXO7 protein (Novus Biologicals, H00025793-P01) or GST protein was mixed with recombinant human His-MBP-PRMT1 protein (Novus Biologicals, NBC1-18446) in NP-40 buffer at 4 °C overnight.

Techniques: Co-Immunoprecipitation Assay, Negative Control, Western Blot, Recombinant, Incubation, Immunoprecipitation

Journal: Nature Communications

Article Title: FBXO7 ubiquitinates PRMT1 to suppress serine synthesis and tumor growth in hepatocellular carcinoma

doi: 10.1038/s41467-024-49087-2

Figure Lengend Snippet: a Immunoblotting analysis of PRMT1 (anti-PRMT1 antibody: Abcam, ab190892) and FBXO7 in FBXO7 KD cells treated with or without MG132 (25 μM, 6 h). The immunoblotting experiments were repeated three times with similar results. b Immunoblotting analysis of PRMT1 and GFP-FBXO7 in FBXO7-overexpressing cells treated with or without MG132 (25 μM) for 6 h. The immunoblotting experiments were repeated three times with similar results. c Immunoblotting analysis of PRMT1 and FBXO7 in FBXO7 KD cells treated with or without cycloheximide (CHX, 50 μg/mL) for the indicated time. Quantitation of PRMT1 protein level based on band intensity was shown (bottom). Data are presented as the mean ± SD ( n = 3 independent experiments with similar results). Statistical analysis was performed using the two-way ANOVA with Bonferroni correction. d Schematic representation of full-length (FL) FBXO7 and enzymatically dead mutant (deletion of F-box domain, shown as ΔF-box). e GFP-PRMT1 and HA-ubiquitin (HA-Ub) were co-expressed with FLAG-FBXO7 WT or enzymatically dead ΔF-box mutant in Huh7 cells. After MG132 (25 μM, 6 h) treatment, IP was performed with GFP antibody, followed by immunoblotting with indicated antibodies. The immunoblotting experiments were repeated three times with similar results. f GFP-PRMT1 was co-expressed with HA-Ub in FBXO7 KD cells. After MG132 (25 μM, 6 h) treatment, IP was performed with GFP antibody, followed by immunoblotting with indicated antibodies. The immunoblotting experiments were repeated three times with similar results. g FBXO7 KD cells were transfected with HA-Ub plasmid and treated with MG132 (25 μM, 6 h). IP was performed with IgG or PRMT1 antibody, followed by immunoblotting with indicated antibodies. The immunoblotting experiments were repeated three times with similar results. Source data are provided as a Source Data file.

Article Snippet: Recombinant human GST-FBXO7 protein (Novus Biologicals, H00025793-P01) or GST protein was mixed with recombinant human His-MBP-PRMT1 protein (Novus Biologicals, NBC1-18446) in NP-40 buffer at 4 °C overnight.

Techniques: Western Blot, Quantitation Assay, Mutagenesis, Transfection, Plasmid Preparation

Journal: Nature Communications

Article Title: FBXO7 ubiquitinates PRMT1 to suppress serine synthesis and tumor growth in hepatocellular carcinoma

doi: 10.1038/s41467-024-49087-2

Figure Lengend Snippet: a Schematic of the workflow for identifying the ubiquitination sites of PRMT1 by mass spectrometry analysis. b Four potential lysine ubiquitination sites (K37, K82, K202, K325) in PRMT1 identified by mass spectrometry analysis. c GFP-PRMT1 (WT or indicated KR mutants) was co-expressed with HA-Ub in FBXO7 KD Huh7 cells rescued with or without FLAG-FBXO7. After MG132 (25 μM, 6 h) treatment, IP was performed with GFP antibody, followed by immunoblotting with indicated antibodies. The immunoblotting experiments were repeated three times with similar results. d FLAG-FBXO7 was co-expressed with GFP-PRMT1 WT or K37R in Huh7 and PLC/PRF/5 cells, followed by immunoblotting with indicated antibodies. The immunoblotting experiments were repeated three times with similar results. e FBXO7 KD cells were transfected with GFP-PRMT1 WT or K37R plasmid, and treated with or without cycloheximide (CHX, 50 μg/mL) for the indicated time. Immunoblotting analysis of GFP-PRMT1 and FBXO7 was performed. Quantitation of GFP-PRMT1 protein level based on band intensity was shown (bottom). Data are presented as the mean ± SD ( n = 3 independent experiments with similar results). Statistical analysis was performed using the two-way ANOVA with Bonferroni correction. f Mass spectrometry identification of K37 ubiquitination of PRMT1. All immunoblotting experiments were repeated three times with similar results. Source data are provided as a Source Data file.

Article Snippet: Recombinant human GST-FBXO7 protein (Novus Biologicals, H00025793-P01) or GST protein was mixed with recombinant human His-MBP-PRMT1 protein (Novus Biologicals, NBC1-18446) in NP-40 buffer at 4 °C overnight.

Techniques: Mass Spectrometry, Western Blot, Transfection, Plasmid Preparation, Quantitation Assay

Journal: Nature Communications

Article Title: FBXO7 ubiquitinates PRMT1 to suppress serine synthesis and tumor growth in hepatocellular carcinoma

doi: 10.1038/s41467-024-49087-2

Figure Lengend Snippet: a , b PHGDH was immunoprecipitated in FBXO7 KD ( a ) or GFP-FBXO7-overexpressing ( b ) cells, followed by immunoblotting with indicated antibodies. The immunoblotting experiments were repeated three times with similar results. c PHGDH was immunoprecipitated in FBXO7 and/or PRMT1 KD cells, followed by immunoblotting with indicated antibodies. The immunoblotting experiments were repeated three times with similar results. d PHGDH was immunoprecipitated in cells overexpressing FLAG-FBXO7 and GFP-PRMT1-WT or K37R, followed by immunoblotting with indicated antibodies. The immunoblotting experiments were repeated three times with similar results. e Endogenous PHGDH was immunoprecipitated in HEK293T, Huh7, and PLC/PRF/5 cells with FBXO7 KD, followed by measurement of PHGDH activity. Data are presented as the mean ± SD ( n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t -test. f GFP-FBXO7 plasmid was transfected in HEK293T cells stably expressing FLAG-PHGDH, followed by IP with FLAG beads and elution with FLAG peptides. PHGDH activity was then measured. Data are presented as the mean ± SD ( n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t -test. g Endogenous PHGDH was immunoprecipitated in FBXO7 and/or PRMT1 KD cells, followed by measurement of PHGDH activity. Data are presented as the mean ± SD ( n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t -test. h Endogenous PHGDH was immunoprecipitated in cells overexpressing FLAG-FBXO7 and GFP-PRMT1-WT or K37R, followed by measurement of PHGDH activity. Data are presented as the mean ± SD ( n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t -test. Source data are provided as a Source Data file.

Article Snippet: Recombinant human GST-FBXO7 protein (Novus Biologicals, H00025793-P01) or GST protein was mixed with recombinant human His-MBP-PRMT1 protein (Novus Biologicals, NBC1-18446) in NP-40 buffer at 4 °C overnight.

Techniques: Immunoprecipitation, Western Blot, Activity Assay, Two Tailed Test, Plasmid Preparation, Transfection, Stable Transfection, Expressing

Journal: Nature Communications

Article Title: FBXO7 ubiquitinates PRMT1 to suppress serine synthesis and tumor growth in hepatocellular carcinoma

doi: 10.1038/s41467-024-49087-2

Figure Lengend Snippet: a Schematic of U-[ 13 C]-glucose incorporation into serine and glycine in cells. b, c Incorporation of U-[ 13 C]-glucose carbon into serine ( b ) and glycine ( c ) detected by LC–MS/MS in cells overexpressing FLAG-FBXO7 and GFP-PRMT1-WT or K37R. Data are presented as the mean ± SD ( n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t -test. d, e GSH ( d ) and ROS ( e ) levels in FBXO7 and/or PRMT1 KD cells cultured in CM or -SG medium. Data are presented as the mean ± SD ( n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t -test. f ROS levels in cells overexpressing FLAG-FBXO7 and GFP-PRMT1-WT or K37R cultured in CM or -SG medium. Data are presented as the mean ± SD ( n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t -test. g ROS levels in FLAG-FBXO7-overexpressing cells cultured in -SG medium in the presence or absence of NAC (5 mM). Data are presented as the mean ± SD ( n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t -test. Source data are provided as a Source Data file.

Article Snippet: Recombinant human GST-FBXO7 protein (Novus Biologicals, H00025793-P01) or GST protein was mixed with recombinant human His-MBP-PRMT1 protein (Novus Biologicals, NBC1-18446) in NP-40 buffer at 4 °C overnight.

Techniques: Liquid Chromatography with Mass Spectroscopy, Two Tailed Test, Cell Culture

Journal: Nature Communications

Article Title: FBXO7 ubiquitinates PRMT1 to suppress serine synthesis and tumor growth in hepatocellular carcinoma

doi: 10.1038/s41467-024-49087-2

Figure Lengend Snippet: a Growth rates of FBXO7 and/or PRMT1 KD cells grown in CM or −SG medium. Data are presented as the mean ± SD ( n = 5 independent experiments). Statistical analysis was performed using the two-way ANOVA with Bonferroni correction. b Growth rates of cells overexpressing FLAG-FBXO7 and GFP-PRMT1-WT or K37R grown in CM or −SG medium. Data are presented as the mean ± SD ( n = 5 independent experiments). Statistical analysis was performed using the two-way ANOVA with Bonferroni correction. c Growth rates of FLAG-FBXO7-overexpressing cells grown in -SG medium in the presence or absence of NAC (5 mM). Data are presented as the mean ± SD ( n = 5 independent experiments). Statistical analysis was performed using the two-way ANOVA with Bonferroni correction. d Immunoblotting analysis of cleaved caspase 3 and caspase 3 in FLAG-FBXO7-overexpressing cells grown in −SG medium in the presence or absence of NAC (5 mM). The immunoblotting experiments were repeated three times with similar results. e , f FBXO7 and/or PRMT1 KD Huh7 cells were subcutaneously inoculated into nude mice fed with a +SG or −SG diet. Tumor image ( e ) and weight ( f ) were shown. Data are presented as the mean ± SD ( n = 6 mice). Statistical analysis in f was performed using the two-tailed Student’s t -test. g , h Huh7 cells overexpressing FLAG-FBXO7 and GFP-PRMT1-WT or K37R were subcutaneously inoculated into nude mice fed with a +SG or −SG diet. Tumor image ( g ) and weight ( h ) were shown. Data are presented as the mean ± SD ( n = 5 mice). Statistical analysis in h was performed using the two-tailed Student’s t -test. i Representative images of IHC staining for Ki-67, 8-oxo-dG, and cleaved caspase 3 in tumor xenografts in ( g ). Scale bars, 50 μm. Source data are provided as a Source Data file.

Article Snippet: Recombinant human GST-FBXO7 protein (Novus Biologicals, H00025793-P01) or GST protein was mixed with recombinant human His-MBP-PRMT1 protein (Novus Biologicals, NBC1-18446) in NP-40 buffer at 4 °C overnight.

Techniques: Western Blot, Two Tailed Test, Immunohistochemistry

Journal: Nature Communications

Article Title: FBXO7 ubiquitinates PRMT1 to suppress serine synthesis and tumor growth in hepatocellular carcinoma

doi: 10.1038/s41467-024-49087-2

Figure Lengend Snippet: a , b Representative images ( a ) and quantitative analysis ( b ) of IHC staining for FBXO7 in HCC tissues and paired normal tissues ( n = 45 samples). Scale bars, 50 μm. Statistical analysis was performed using the paired two-tailed Student’s t -test. c , d Representative images ( c ) and quantitative analysis ( d ) of IHC staining for PRMT1 in HCC tissues and paired normal tissues ( n = 45 samples). Scale bars, 50 μm. Statistical analysis was performed using the paired two-tailed Student’s t -test. e and f Representative images ( e ) and quantitative analysis ( f ) of IHC staining for mePHGDH (R236me1) in HCC tissues and paired normal tissues ( n = 45 samples). Scale bars, 50 μm. Statistical analysis was performed using the paired two-tailed Student’s t -test. g Representative images of IHC staining with FBXO7, PRMT1, and mePHGDH (R236me1) antibodies in HCC tissues ( n = 45 samples). Scale bars, 50 μm. h Two-tailed Pearson correlation test analyzing the relationship between the IHC staining intensity of FBXO7 and PRMT1 in HCC tissues ( n = 45 samples). i Two-tailed Pearson correlation test analyzing the relationship between the IHC staining intensity of FBXO7 and mePHGDH (R236me1) in HCC tissues ( n = 45 samples). j Two-tailed Pearson correlation test analyzing the relationship between the IHC staining intensity of PRMT1 and mePHGDH (R236me1) in HCC tissues ( n = 45 samples). k IP and immunoblotting analysis with indicated antibodies in 6 HCC tissues (T1–T6). The immunoblotting experiments were repeated three times with similar results. l Two-tailed Pearson correlation test analyzing the relationship between the levels of FBXO7–PRMT1 interaction and PRMT ubiquitination in HCC tissues based on band intensity in k ( n = 6 samples). m Schematic model for the mechanism of FBXO7–PRMT1–PHGDH axis in regulating serine synthesis, oxidative stress, and HCC growth. Source data are provided as a Source Data file.

Article Snippet: Recombinant human GST-FBXO7 protein (Novus Biologicals, H00025793-P01) or GST protein was mixed with recombinant human His-MBP-PRMT1 protein (Novus Biologicals, NBC1-18446) in NP-40 buffer at 4 °C overnight.

Techniques: Immunohistochemistry, Two Tailed Test, Western Blot

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