Journal: Journal of proteome research

Article Title: Combined Antibody/Lectin-Enrichment Identifies Extensive Changes in the O -GlcNAc Sub-proteome Upon Oxidative Stress

doi: 10.1021/acs.jproteome.6b00369

Figure Lengend Snippet: (A) OGT wild-type and null cells were treated with hydrogen peroxide (2.5mM; 1–3h). Proteins were enriched from nuclear and cytoplasmic lysates using the G5-lectibody column and eluted with 1M GlcNAc. 4% of the input or 20% of the eluate were separated by SDS-PAGE and the following proteins were detected by immunoblot: O-GlcNAc, OGT, hnRNP-U, Caprin, Carm1, PRMT1, VDAC, PRMT5, NFκB p65, NFκB p50, Fus, Actin, Nup62, 14–3-3 phospho-substrate, 14–3-3γ, and 14–3-3η. (B) The SILAC ratios for the proteins tested in Figure 5a are reported. (C) OGT, Carm1, hnRNP-U, 14–3-3 proteins, Caprin, and Fus, were immunoprecipitated from Control, Stressed cells (2.5 mM H2O2, 1–3 h) and OGT null cells. The levels of O-GlcNAc on the indicated proteins were assessed by immunoblot with CTD110.6.

Article Snippet: Antibodies used in this study include: OGT (DM-17), heterogeneous Nuclear Ribonucleoprotein U (hnRNP-U) and actin (Sigma-Aldrich); Carm1 (coactivator-associated arginine methyltransferase 1), Caprin and FUS RNA binding protein (Fus; Bethyl Laboratories); heat shock cognate 71 kDa (Hsc70), Protein Arginine Methyltransferase 1 (PRMT1), Voltage-dependent anion channels (VDAC), Protein Arginine Methyltransferase 5 (PRMT5), nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), and Casein kinase 2 (CK2; Santa Cruz Biotechnology); AKT phospho-substrate, 14–3-3 substrate, 14–3-3γ, 14–3-3η (Cell Signaling); and mAb414 (Abcam).

Techniques: SDS Page, Western Blot, Immunoprecipitation

Journal: Journal of proteome research

Article Title: Combined Antibody/Lectin-Enrichment Identifies Extensive Changes in the O -GlcNAc Sub-proteome Upon Oxidative Stress

doi: 10.1021/acs.jproteome.6b00369

Figure Lengend Snippet: (A) Mouse embryonic fibroblasts (MEFs) were labeled with medium and heavy isotopes of arginine and lysine, and treated with 2.5 mM H2O2 for 1 and 2 h respectively. Equal amounts of protein were combined and O-GlcNAc-modified proteins were purified using a combination of immobilized O-GlcNAc-specific antibodies (CTD110.6, RL2, HGAC39 and HGAC89) and WGA referred to as the G5-lectibody resin/column. Eluted proteins were trypsin digested, separated into 12 fractions by basic reversed phase (bRP) chromatography and subjected to LC-MS/MS. (B) To reduce antibody contamination of the eluent, the G5-lectibody resin was packed on top of a trap column containing equal parts Protein A, Protein G and anti-IgM sepharose. (C) O-GlcNAc modified proteins were enriched from Nuc/Cyt lysates using sepharose-4B covalently coupled to the indicated antibodies and lectins at 2mg/ml. Immunoprecipitates were separated by SDS-PAGE and OGT, OGA, Carm1, Nup62, Caprin, hnRNP-U and Casein Kinase 2 α (CK2α) were detected by immunoblot. (D) O-GlcNAc modified proteins were enriched from wildtype (WT) or OGT null (KO) Nuc/Cyt lysates (400 μg) or buffer alone (B) using WGA sepharose (2.5 or 10 nmol of GlcNAc binding) and the G5-lectibody column (3 nmoles GlcNAc-binding). Input (5μg, 1.4%), unbound (5μg, 1.4%), and immunoprecipitated (5.25%) proteins were separated by SDS-PAGE. OGT, Carm1, and Caprin were detected by immunoblot (n=3).

Article Snippet: Antibodies used in this study include: OGT (DM-17), heterogeneous Nuclear Ribonucleoprotein U (hnRNP-U) and actin (Sigma-Aldrich); Carm1 (coactivator-associated arginine methyltransferase 1), Caprin and FUS RNA binding protein (Fus; Bethyl Laboratories); heat shock cognate 71 kDa (Hsc70), Protein Arginine Methyltransferase 1 (PRMT1), Voltage-dependent anion channels (VDAC), Protein Arginine Methyltransferase 5 (PRMT5), nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), and Casein kinase 2 (CK2; Santa Cruz Biotechnology); AKT phospho-substrate, 14–3-3 substrate, 14–3-3γ, 14–3-3η (Cell Signaling); and mAb414 (Abcam).

Techniques: Labeling, Modification, Purification, Chromatography, Liquid Chromatography with Mass Spectroscopy, SDS Page, Western Blot, Binding Assay, Immunoprecipitation

Journal: Theranostics

Article Title: TFE3, a potential therapeutic target for Spinal Cord Injury via augmenting autophagy flux and alleviating ER stress

doi: 10.7150/thno.46566

Figure Lengend Snippet: The activity of TFE3 after SCI is regulated by AMPK-mTOR and AMPK-SKP2-CARM1 signaling pathways. ( A ) Western blot analysis of AMPK-mTOR signal pathway in the cytoplasm in spinal cords from the control mice, SCI mice, and SCI mice treated with DMSO or Compound C, at Day3. ( B ) Densitometric analysis of the AMPK, p-AMPK, p-mTOR and p-4EBP1 data from (A), normalized to the loading control GAPDH. ( C ) Western blotting of TFE3 nuclear translocation at the lesion for each group. ( D ) Corresponding densitometric analysis of the TFE3 bands in (C) normalized to the loading control H3. ( E ) Western blots of the AMPK-SKP2-CARM1 signaling pathway in the nucleus of the indicated groups at Day3 after SCI. ( F ) Densitometric analysis of AMPK, p-AMPK, p-FOXO3a, SKP2 and CARM1 bands from (E) normalized to control H3. ( G ) Nuclear CARM1-TFE3 complex was detected by IP in indicated groups at Day3 after SCI. ( H ) Densitometric analysis of TFE3 and CARM1 data from (G) normalized to loading control H3. ( I ) Western blot analysis of LC3, SQSTM1/p62 and UB in the spinal cord lesion of each group at Day3 after SCI. ( J ) Densitometric analysis of band data from (I) normalized to the loading control GAPDH. ( K ) Western blotting of LC3II in the indicated mice spinal cord slides cultured in the presence or absence of CQ at Day3. ( L ) Densitometric analysis of LC3II from (K) normalized to the loading control GAPDH. n=6, ns stands for not significant, *P<0.05, **P<0.01.

Article Snippet: Primary antibodies against Beclin1 (Cat. No. 3738), ATG5 (Cat. No. 12994), ATP6V1B2 (Cat. No. 14617), Ubiquitin (Cat. No. 3936), ATF4 (Cat. No. 11815), CHOP (Cat. No. 2895), AMPKα (Cat. No. 5832), p-AMPKα (Cat. No. 2535) p-FOXO3a (Cat. No. 9466), p-EIF2α (Cat. No. 3398), p-4EBP1 (Cat. No. 9456), mTOR (Cat. No.2983), p-mTOR (Cat. No.5536) and CARM1 (Cat. No. 4438) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activity Assay, Protein-Protein interactions, Western Blot, Control, Translocation Assay, Cell Culture

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Inhibition of CARM1-Mediated Methylation of ACSL4 Promotes Ferroptosis in Colorectal Cancer.

doi: 10.1002/advs.202303484

Figure Lengend Snippet: Figure 1. CARM1 was negatively associated with ferroptosis. a) HE staining of clinical specimens of colon cancer. Scale bar, 20 μm. b) Twenty-five pairs of colorectal cancer (CRC) tissues and adjacent tissues were digested into single-cell suspensions, and lipid ROS production was assayed via flow cytometry by using C11-BODIPY after RSL3 treatment for 4 h (n = 25). c) Malondialdehyde (MDA) levels were detected by using a lipid peroxidation MDA assay kit in single-cell suspensions treated with RSL3 for 4 h from 25 pairs of CRC tissues and adjacent tissues (n = 25). d) Heatmap of RNA-seq using six patient tumors with different lipid ROS levels showing changes in gene expression, including CARM1. e) Quantitative real-time PCR (qPCR) analysis of CARM1 mRNA levels in tumors from 10 patients. f) Western blot analysis of CARM1 in the same tissues as (e). g) Cell viability was assayed in vector- and CARM1-overexpressing LoVo and HCT116 cells treated with the indicated doses of RSL3 and erastin for 24 h. h) Scatter plot of the immunohistochemistry (IHC) staining score for CARM1, lipid ROS, and MDA levels in CRC tissues (n = 25). All p values and R values were calculated with Spearman’s r test. i) Representative results of immunohistochemical staining for CARM1 from 25 clinical CRC patients. Scale bars, 20 μm. j,k) Lipid ROS (left) and MDA (right) levels were compared in CARM1 high (CARM1 IHC score ≥6) and CARM1 low (CARM1 IHC score<6) groups. The data shown represent the mean ± SD. In (b) and (c), comparisons were made by using paired Student’s t-test, and in (j) and (k), comparisons were made by using the two-tailed, unpaired Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: GST Pull-Down Assay: The proteins for CARM1 (OriGene, TP317483) and ACSL4 (H00002182-P01, Abnova) were commercially available.

Techniques: Staining, Cytometry, Multiple Displacement Amplification, RNA Sequencing, Gene Expression, Real-time Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Immunohistochemistry, Immunohistochemical staining, Two Tailed Test

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Inhibition of CARM1-Mediated Methylation of ACSL4 Promotes Ferroptosis in Colorectal Cancer.

doi: 10.1002/advs.202303484

Figure Lengend Snippet: Figure 2. CARM1-KD enhances ferroptotic cell death. a) Cell viability was measured in siNC and siCARM1 LoVo cells treated with 2.5 × 10−6 m RSL3 or 5 × 10−6 m erastin for 12 h (n = 5 independent experiments). b) Cell viability was measured in siNC and siCARM1 LoVo cells treated with cell death inhibitors and 2. 5 × 10−6 m RSL3 or 5 × 10−6 m erastin for 12 h. Fer-1, 1 × 10−6 m ferrostatin-1; NAC, 5 × 10−3 m; Nec, 2 × 10−6 m necrostatin-1; Z-V, 20 × 10−6 m Z-VAD-FMK (n = 5 independent experiments). c,d) Malondialdehyde (MDA) levels and relative lipid ROS were assayed in the indicated LoVo cells treated with 2. 5 × 10−6 m RSL3 or 5 × 10−6 m erastin for 12 h (n = 3 independent experiments). e) Transmission electron microscopy (TEM) images of the indicated LoVo cells subjected to RSL3 (2. 5 × 10−6 m) for 12 h. White arrows indicate mitochondria. Scale bars, left, 2 μm; right, 500 nm. f) The indicated stable LoVo cells were used to evaluate mitochondrial membrane potential via fluorescence staining of mitochondria with JC-1 dye (n = 3 independent experiments). g) shNC and shCARM1 LoVo cells were subcutaneously injected into the mice. RSL3 was administered to all tumors with or without Fer-1. Tumor volumes (n = 5) were calculated every 4 days, and the growth curve was drawn. h) Images of tumors from LoVo xenograft mice with altered treatments are shown, and the tumor weights (n = 5) of the subcutaneous xenografts were measured. i) Representative immunohistochemical images of CARM1 and Ki67 in tumor sections are shown. Scale bars, 20 μm. j,k) MDA levels and relative lipid ROS in tumor cells isolated from (h) were assayed (n = 5 independent experiments). The data shown represent the mean ± SD. Comparisons were made by using one-way ANOVA with Tukey’s test; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: GST Pull-Down Assay: The proteins for CARM1 (OriGene, TP317483) and ACSL4 (H00002182-P01, Abnova) were commercially available.

Techniques: Transmission Assay, Electron Microscopy, Membrane, Staining, Injection, Immunohistochemical staining, Isolation

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Inhibition of CARM1-Mediated Methylation of ACSL4 Promotes Ferroptosis in Colorectal Cancer.

doi: 10.1002/advs.202303484

Figure Lengend Snippet: Figure 4. CARM1 directly interacts with and decreases ACSL4 protein levels in colon cancer cells. a) Mass spectrometry analysis identified ACSL4 in the binding protein pool of CARM1. b) Immunoprecipitation (IP) analyses were performed to examine the endogenous interaction between CARM1 and ACSL4 by using antibodies against CARM1 and ACSL4 in LoVo cells. c) IP analyses were performed to examine the exogenous interaction between CARM1 and ACSL4 by using antibodies against Flag and HA, respectively, in HEK293T cells. d) In vitro GST pull-down assay to verify the binding of CARM1 and ACSL4. e) Immunofluorescence staining was performed to observe the colocalization of CARM1 (green) and ACSL4 (red) in LoVo and

Article Snippet: GST Pull-Down Assay: The proteins for CARM1 (OriGene, TP317483) and ACSL4 (H00002182-P01, Abnova) were commercially available.

Techniques: Mass Spectrometry, Binding Assay, Immunoprecipitation, In Vitro, Pull Down Assay, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Inhibition of CARM1-Mediated Methylation of ACSL4 Promotes Ferroptosis in Colorectal Cancer.

doi: 10.1002/advs.202303484

Figure Lengend Snippet: Figure 5. CARM1 methylates ACSL4 at R339. a,b) Co-immunoprecipitation (Co-IP) was performed to detect the methylation levels of ACSL4 with CARM1 attenuation (left) or upregulation (right). c) IP assay was performed for the enrichment of ACSL4 protein, staining was performed with Coomassie bright

Article Snippet: GST Pull-Down Assay: The proteins for CARM1 (OriGene, TP317483) and ACSL4 (H00002182-P01, Abnova) were commercially available.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Methylation, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Inhibition of CARM1-Mediated Methylation of ACSL4 Promotes Ferroptosis in Colorectal Cancer.

doi: 10.1002/advs.202303484

Figure Lengend Snippet: Figure 7. RNF25 knockdown inhibits CARM1-induced ferroptosis resistance. a) Western blot analysis of LoVo and HCT116 cells transfected with the indicated plasmid and siRNAs. Protein levels of CARM1, RNF25, ACSL4 and ACSL4 R339me2a were assayed. b) Western blot analysis of vector- and RNF25-overexpressing LoVo and HCT116 cells treated with DMSO or 10 × 10−9 m EZM2302 for 24 h. Protein levels of RNF25, ACSL4, and H3R17me2a were assayed. c) HEK293T cells transfected with the indicated plasmids and treated with or without 10 × 10−9 m EZM2302 for 24 h. Immunoprecipitation (IP) with an anti-Flag antibody and Western blotting with an anti-Myc antibody were performed to detect the ubiquitination level of ACSL4. d) Cell viability was assayed in the indicated LoVo and HCT116 cells as (a) treated with 2. 5 × 10−6 m RSL3 for 12 h (n = 5 independent experiments). e,f) Malondialdehyde (MDA) levels and relative lipid ROS were assayed in the indicated LoVo and HCT116 cells treated with 2. 5 × 10−6 m RSL3 for 12 h (n = 3 independent experiments). g) Mitochondrial membrane potential was detected for the same cells as (e) by using fluorescence staining of mitochondria with JC-1 dye (n = 3 independent experiments). h) Schematic diagram of our hypothesis about this project. The data shown represent the mean ± SD. Comparisons were made by using one-way ANOVA with Tukey’s test; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: GST Pull-Down Assay: The proteins for CARM1 (OriGene, TP317483) and ACSL4 (H00002182-P01, Abnova) were commercially available.

Techniques: Knockdown, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Ubiquitin Proteomics, Membrane, Staining

Journal: EMBO Reports

Article Title: The L27 domain of MPP7 enhances TAZ-YY1 cooperation to renew muscle stem cells

doi: 10.1038/s44319-024-00305-4

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Myc-Carm1 (human cDNA) , Origene , Catalog #RC217483.

Techniques: Recombinant, Modification, Magnetic Beads, Sequencing, Blocking Assay, Plasmid Preparation, Electron Microscopy, Software, Reporter Assay, Imaging, In Situ

Journal: iScience

Article Title: Cooperation between PRMT1 and PRMT6 drives lung cancer health disparities among Black/African American men

doi: 10.1016/j.isci.2024.108858

Figure Lengend Snippet: Expression of PRMT1, PRMT4, and PRMT6 is elevated in NSCLC tissue, and higher PRMT6 levels are detected in LUAD tissue of Black/AA men compared to NHW men (A) Extracts of uninvolved lung tissue and tumor tissue from the same patient were analyzed by immunoblotting using anti-PRMT1, anti-PRMT4, anti-PRMT6, or anti-vinculin. A portion of Ponceau S staining for equal protein loading is shown. (B) Quantification of signals in (A) is shown. Bars indicate the mean of all samples (two-tailed t-test for p value). (C) Extracts of LUAD tissue from NHW men and Black/AA men were analyzed by immunoblotting using anti-PRMT1, anti-PRMT4, anti-PRMT6, or anti-vinculin. A portion of Ponceau S staining for equal protein loading is shown. Vinculin blots here are the same ones in Figure S6 D. (D) Quantification of signals in (C) is shown. Bars indicate the mean of all samples (two-tailed t-test for p value). (E and F) mRNA levels of PRMT1 (E) and PRMT6 (F) in LUAD tissue from NHW men and Black/AA men were analyzed by qRT-PCR. Bars indicate the mean of all samples (two-tailed t-test for p value). See also Figures S1 and .

Article Snippet: A plasmid expressing HA-tagged PRMT4 was purchased from Addgene (#81118).

Techniques: Expressing, Western Blot, Staining, Two Tailed Test, Quantitative RT-PCR

Journal: iScience

Article Title: Cooperation between PRMT1 and PRMT6 drives lung cancer health disparities among Black/African American men

doi: 10.1016/j.isci.2024.108858

Figure Lengend Snippet: PRMT1 interacts with PRMT6 (A) H1299 cells were co-transfected with a control vector or a vector expressing FLAG-PRMT6 and vectors expressing HA-PRMT1 or HA-PRMT4. Cell extracts were subjected to anti-FLAG immunoprecipitation. The immunoprecipitates were analyzed by anti-HA or anti-FLAG immunoblotting. (B) H1299 cells were transfected with a control vector or a vector expressing FLAG-PRMT6. Cell extracts were subjected to anti-FLAG immunoprecipitation. The immunoprecipitates were analyzed by anti-PRMT1, anti-PRMT4, or anti-FLAG immunoblotting. (C) GST or GST-PRMT6 was incubated with His-PRMT1. The reactions were subjected to GST pulldown followed by anti-PRMT1 or anti-GST immunoblotting. (D) His-PRMT1 incubated with GST-PRMT6 was subjected to Ni-NTA pulldown followed by anti-PRMT6 or anti-PRMT1 immunoblotting. (E) H1299 cells were co-transfected with vectors expressing YFPn-PRMT1 and YFPc-PRMT1, YFPn-PRMT6 and YFPc-PRMT6, YFPn-PRMT6 and YFPc-PRMT1, YFPn-PRMT1 and YFPc-MS2, or YFPn-PRMT6 and YFPc-MS2. YFP signal was analyzed by fluorescence microscopy. Scale bar, 25 μm. (F) Expression of transfected proteins in (E) was analyzed by anti-FLAG or anti-HA immunoblotting. (G) Extracts of H1299 or PRMT1 KO cells were subjected to anti-PRMT6 immunoprecipitation. The immunoprecipitates were analyzed by anti-PRMT1 or anti-PRMT6 immunoblotting.

Article Snippet: A plasmid expressing HA-tagged PRMT4 was purchased from Addgene (#81118).

Techniques: Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Western Blot, Incubation, Fluorescence, Microscopy

Journal: iScience

Article Title: Cooperation between PRMT1 and PRMT6 drives lung cancer health disparities among Black/African American men

doi: 10.1016/j.isci.2024.108858

Figure Lengend Snippet:

Article Snippet: A plasmid expressing HA-tagged PRMT4 was purchased from Addgene (#81118).

Techniques: Purification, Transduction, Recombinant, Virus, Polymer, SYBR Green Assay, Viability Assay, Mass Spectrometry, Sequencing, Synthesized, Plasmid Preparation, Software, Membrane, Transfection

Journal: Journal of Virology

Article Title: Coactivator-Associated Arginine Methyltransferase 1 Enhances Transcriptional Activity ofthe Human T-Cell Lymphotropic Virus Type 1 Long Terminal Repeat through Direct Interactionwith Tax

doi: 10.1128/jvi.00186-06

Figure Lengend Snippet: FIG. 2. CARM1 mutant does not influence Tax transcription of the HTLV-1 LTR. (A) HeLa cells were transiently transfected using Ef- fectene transfection reagent (QIAGEN) with reporter construct HTLV-1 LTR-Luc (0.1 g), RSV -Gal (0.05 g), CARM1 WT or MT (0.1 g), or Tax (0.1 g) expression plasmids. At 24 h posttransfection, cells were collected and luciferase activities were measured. Luciferase values were adjusted for transfection efficiency using RSV -Gal. The graph represents the luciferase activity from three independent exper- iments. The standard deviation for the three experiments is included. (B) Western blot analysis was performed for Tax (Tab172) and CARM1 (Upstate).

Article Snippet: The transfection with siRNA for CARM1 (Santa Cruz Biotechnologies) or chloramphenicol acetyltransferase (CAT) (QIAGEN) was performed 12 h prior to DNA transfection using RNAifect transfection reagent (QIAGEN).

Techniques: Mutagenesis, Transfection, Construct, Expressing, Luciferase, Activity Assay, Standard Deviation, Western Blot

Journal: Journal of Virology

Article Title: Coactivator-Associated Arginine Methyltransferase 1 Enhances Transcriptional Activity ofthe Human T-Cell Lymphotropic Virus Type 1 Long Terminal Repeat through Direct Interactionwith Tax

doi: 10.1128/jvi.00186-06

Figure Lengend Snippet: FIG. 3. Blocking endogenous CARM1 using siRNA prevents Tax activation of HTLV-1 LTR. (A) siRNA (100 nM) for CARM1 (Santa Cruz Biotechnologies) or CAT (QIAGEN) was transfected with RNAifect reagent (QIAGEN) into HeLa cells. After 12 h, cells were transiently transfected using Effectene transfection reagent with re- porter construct HTLV-1 LTR-Luc (0.1 g), RSV -Gal (0.05 g), or Tax (0.1 g) expression plasmids. At 24 h posttransfection, cells were collected, and luciferase activities were measured. Luciferase values were adjusted for transfection efficiency using RSV -Gal. The graph represents the luciferase activity from three independent experiments. The standard deviation for the three experiments is included. (B) Western blot analysis was performed for Tax (Tab172) and CARM1 (Upstate).

Article Snippet: The transfection with siRNA for CARM1 (Santa Cruz Biotechnologies) or chloramphenicol acetyltransferase (CAT) (QIAGEN) was performed 12 h prior to DNA transfection using RNAifect transfection reagent (QIAGEN).

Techniques: Blocking Assay, Activation Assay, Transfection, Construct, Expressing, Luciferase, Activity Assay, Standard Deviation, Western Blot

Journal: Journal of Virology

Article Title: Coactivator-Associated Arginine Methyltransferase 1 Enhances Transcriptional Activity ofthe Human T-Cell Lymphotropic Virus Type 1 Long Terminal Repeat through Direct Interactionwith Tax

doi: 10.1128/jvi.00186-06

Figure Lengend Snippet: FIG. 4. CARM1 and Tax directly interact in vitro and in vivo. (A and B) GST or GST-Tax wild type (A, lanes 1 and 2) and GST-Tax deletion mutants (B) were incubated with purified CARM1 protein. Shown is Western blot analysis of CARM1 levels from GST-Tax pull-down after incubation of HeLa cell nuclear extract with GST or GST-Tax (A, top panel, lanes 3 and 4). Samples were separated by electrophoresis in 4 to 20% Tris-glycine gels, and Western blot analysis for CARM1 or GST was performed. (C) Nuclear extracts from C81 cells were immunoprecipitated (IP) with anti-CARM1 antibody (Ab) and washed extensively, and pro- teins were separated by electrophoresis in 4 to 20% Tris-glycine gels. Western blot analyses for Tax, CARM1, or p300 were performed. (D) Co- localization of Tax and CARM1. C81 cells were fixed and then immuno- stained with anti-Tax (-Tax) and/or anti-CARM1 (-CARM1) antibod- ies. (E) 293T cells were transfected with Tax WT or mutant del 151–204, M29, M30, or M32, using Effectene transfection reagent (QIAGEN). At 48 h posttransfection, cell lysates were prepared and immunoprecipated with anti-CARM1 antibody. Immunoprecipitates were separated by electrophore- sis in 4 to 20% Tris-glycine gels. Western blot analyses for Tax or CARM1 were performed. The lower panel indicates the levels of expression of Tax and CARM1. (F) Luciferase activities of HTLV-1 LTR were measured in 293T cells transfected with Tax WT or mutants.

Article Snippet: The transfection with siRNA for CARM1 (Santa Cruz Biotechnologies) or chloramphenicol acetyltransferase (CAT) (QIAGEN) was performed 12 h prior to DNA transfection using RNAifect transfection reagent (QIAGEN).

Techniques: In Vitro, In Vivo, Incubation, Western Blot, Electrophoresis, Immunoprecipitation, Staining, Transfection, Mutagenesis, Expressing, Luciferase

Journal: Journal of Virology

Article Title: Coactivator-Associated Arginine Methyltransferase 1 Enhances Transcriptional Activity ofthe Human T-Cell Lymphotropic Virus Type 1 Long Terminal Repeat through Direct Interactionwith Tax

doi: 10.1128/jvi.00186-06

Figure Lengend Snippet: FIG. 5. CARM1 is recruited to the HTLV-1 PICs in the presence of Tax. HTLV-1 PICs were assembled by incubating biotinylated HTLV-1 templates with HeLa nuclear extracts (ext) in the absence or presence of the His6-Tax WT or mutant (del 151–204) and then puri- fied with streptavidin-coated magnetic beads. The protein components of the purified PICs were analyzed by Western blotting with anti-Tax (A), -CARM1 (B), -CREB (C), or -p300 (D) antibodies. DNA-bio, biotinylated DNA.

Article Snippet: The transfection with siRNA for CARM1 (Santa Cruz Biotechnologies) or chloramphenicol acetyltransferase (CAT) (QIAGEN) was performed 12 h prior to DNA transfection using RNAifect transfection reagent (QIAGEN).

Techniques: Mutagenesis, Magnetic Beads, Western Blot

Journal: Journal of Virology

Article Title: Coactivator-Associated Arginine Methyltransferase 1 Enhances Transcriptional Activity ofthe Human T-Cell Lymphotropic Virus Type 1 Long Terminal Repeat through Direct Interactionwith Tax

doi: 10.1128/jvi.00186-06

Figure Lengend Snippet: FIG. 6. CARM1 is associated with active HTLV-1 LTR chromatin in vivo. SP cells, which contain a single active integrated copy of the HTLV-1 proviral genome, were subjected to ChIP assays. Antibodies specific for Tax, CARM1, dimethyl histone H3 (R2, R17, R26, or K9), histone H3, and acetylated histone H3 (K9) were used for immuno- precipitation. PCRs were carried out to analyze precipitated DNA using primers specific for the HTLV-1 LTR and -globin promoter region.

Article Snippet: The transfection with siRNA for CARM1 (Santa Cruz Biotechnologies) or chloramphenicol acetyltransferase (CAT) (QIAGEN) was performed 12 h prior to DNA transfection using RNAifect transfection reagent (QIAGEN).

Techniques: In Vivo, Immunoprecipitation

Journal: iScience

Article Title: PRMT4-mediated arginine methylation promotes tyrosine phosphorylation of VEGFR-2 and regulates filopodia protrusions

doi: 10.1016/j.isci.2022.104736

Figure Lengend Snippet:

Article Snippet: PRMT4/CRAM1 , SCBT , Human PRMT4/CRAM1 shRNA (Cat # sc-44875-SH).

Techniques: Recombinant, shRNA, Software