Journal: Cell

Article Title: Mapping Systemic Inflammation and Antibody Responses in Multisystem Inflammatory Syndrome in Children (MIS-C)

doi: 10.1016/j.cell.2020.09.034

Figure Lengend Snippet:

Article Snippet: anti-GranzymeB 111Cd-conjugated Clone REA226 , Miltenyi , Cat No.130-108-055; RRID: AB_2659980.

Techniques: Virus, Recombinant, Saline, Blocking Assay, Staining, Microarray, Software, Immunoprecipitation

Journal: Cell reports

Article Title: Activation of miR-21-Regulated Pathways in Immune Aging Selects against Signatures Characteristic of Memory T Cells

doi: 10.1016/j.celrep.2018.10.074

Figure Lengend Snippet: (A) Naive CD4 + T cells were transfected with either scrambled control or LNA21. Representative western blots and mean normalized band intensities of PTEN, SPRY1, and b-actin expression after 48 hr are shown (n = 3–4, mean ± SEM). (B and C) Naive CD4 + T cells from young and older individuals were activated with anti-CD3 and anti-CD28 beads and transduced with a lentiviral vector expressing scrambled control RNA or anti-miR-21. (B) Representative histogram of phosphorylated S6 in GFP + cells on day 2 and results from 7 individuals. (C) Representative histograms of phosphorylated S6, AKT, mTOR, and ERK in GFP + cells on day 3 and results of paired samples from 7 individuals. The filled gray histograms represent unstimulated naive CD4 + T cells. (D and E) Naive CD4 + T cells from healthy adults were activated with anti-CD3 and anti-CD28 beads. On day 3, activated cells were treated with combinations of the AKT inhibitor MK-2206 2HCl and the MEK1 and MEK2 inhibitor U0126 for 1.5 hr. (D) Representative histograms show phosphorylated S6, mTOR, and ERK in cells treated with combinations of 40 nM of the AKT inhibitor and 400 nM of MEK1 and MEK2 inhibitor. Numbers in histograms indicate geometric MFI of p-mTOR and p-ERK. (E) Graphs depicting the frequencies of cells with phosphorylated S6 cultured with the indicated concentrations of the AKT inhibitor in the presence or absence of 400 nM of the MEK1 and MEK2 inhibitor (n = 4, mean ± SEM). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; all by two-tailed paired t test.

Article Snippet: p-mTOR (S2448; O21-404) , BD Biosciences , Cat# 564242; RRID: AB_2738695.

Techniques: Transfection, Western Blot, Expressing, Transduction, Plasmid Preparation, Cell Culture, Two Tailed Test

Journal: Cell reports

Article Title: Activation of miR-21-Regulated Pathways in Immune Aging Selects against Signatures Characteristic of Memory T Cells

doi: 10.1016/j.celrep.2018.10.074

Figure Lengend Snippet:

Article Snippet: p-mTOR (S2448; O21-404) , BD Biosciences , Cat# 564242; RRID: AB_2738695.

Techniques: Plasmid Preparation, Recombinant, Isolation, SYBR Green Assay, Reporter Assay, Microarray, Luciferase, Software

Journal: Autophagy

Article Title: CARM1 drives mitophagy and autophagy flux during fasting-induced skeletal muscle atrophy.

doi: 10.1080/15548627.2023.2288528

Figure Lengend Snippet: Figure 1. Fasting-induced atrophy in carm1 mKO (skeletal muscle-specific knockout) mice. (A) Representative western blots of CARM1 protein content (normal and long exposures) in the TA (tibialis anterior) muscle from WT (wild-type) and mKO mice under fed and fast (24 and 48 h) conditions, as well as a representative Ponceau stain, below. Molecular masses (kDa) are shown at right of blots. (B) graphical summary of CARM1 protein expression in TA muscle of WT animals. Data are expressed as protein content relative to fed (n = 14). (C) body mass of WT and mKO animals in response to fasting (n = 11–21). (D) TA, quadricep (QUAD), and gastrocnemius (GAST) muscle mass from WT and mKO mice in fed and fast conditions. Muscle weight data are expressed relative to WT fed (n = 11–21). (E) Representative images of hematoxylin and eosin (H&E)-stained EDL muscle cross sections from WT and mKO mice in fed and fast cohorts. Scale bar: 50 μm. (F) graphical summary of the average myofiber cross-sectional area (CSA) of EDL muscles from WT and mKO mice in response to fasting. Data are expressed as CSA relative to the WT fed (n = 8–9). Data are means ± SEM. Two-way ANOVA; $ p < 0.05 interaction effect of genotype and fasting; ¢ p < 0.05 main effect of genotype; ‡

Article Snippet: We employed antibodies against CARM1 (1:5,000; Bethyl Laboratories, A300-421A), PRMT1 (1:1,000; EMD Millipore, 07– 404), PRMT5 (1:1,000; EMD Millipore, 07–405), PRMT6 (1:1,000; Bethyl Laboratories, A300-929A), PRMT7 (1:1,000; Santa Cruz Biotechnology, sc -376,077), MMA (1:1,000; Cell Signaling Technology, 8015S), ADMA (1:1,000; 13522S; Cell Signaling Technology, 13522S), CARM1 substrate (1:1,000; another gift from Dr. Mark Bedford, MD Anderson Cancer Center, University of Texas), SDMA (1:1,000; Cell Signaling Technology, 13222S), asymmetrically dimethylated SMARCC1 me2a (Arg1064; 1:1,000; Cell Signaling Technology, 94962S), SMARCC1 (1:1,000; Cell Signaling Technology, 11956S), asymmetrically dimethylated PABPC1 (Arg455/Arg460; 1:1,000; Cell Signaling Technology, 3505S), and PABPC1 (1:1,000; Cell Signaling Technology, 4992S) to assess PRMT expression and function.

Techniques: Knock-Out, Western Blot, Staining, Expressing, Muscles

Journal: Autophagy

Article Title: CARM1 drives mitophagy and autophagy flux during fasting-induced skeletal muscle atrophy.

doi: 10.1080/15548627.2023.2288528

Figure Lengend Snippet: Figure 2. PRMT (protein arginine methyltransferase) content and activity in mKO animals following fasting-evoked muscle wasting. (A) heatmap of PRMT family members in TA muscle of WT and mKO mice after fasting. Data are expressed relative to WT fed (n = 3). (B) PRMT1, PRMT5, PRMT6, and PRMT7 mRNA expression in EDL muscles from WT and mKO mice during fed and fast conditions. Data are expressed as mRNA content relative to WT fed muscle (n = 9–13). (C) typical western blots of PRMT1, PRMT5, PRMT6, PRMT7, monomethylarginine (MMA), asymmetric dimethylarginine (ADMA), asymmetric arginine dimethylated CARM1 substrates, symmetric dimethylarginine (SDMA), asymmetrically dimethylated SMARCC1/BAF155 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily c, member 1) me2a (Arg1064), total SMARCC1, asymmetrically dimethylated PABPC1/PABP1 (poly(A) binding protein, cytoplasmic 1) me2a (Arg455/ Arg460), and total PABPC1 in TA muscles from WT and mKO animals in fed and fast cohorts, accompanied by a typical Ponceau stain. Molecular masses (kDa) are shown at the right of blots. (D-G) graphical summaries of PRMT1, PRMT5, PRMT6, PRMT7, MMA, ADMA, CARM1 substrate, SDMA, SMARCC1 me2a (Arg1064),

Article Snippet: We employed antibodies against CARM1 (1:5,000; Bethyl Laboratories, A300-421A), PRMT1 (1:1,000; EMD Millipore, 07– 404), PRMT5 (1:1,000; EMD Millipore, 07–405), PRMT6 (1:1,000; Bethyl Laboratories, A300-929A), PRMT7 (1:1,000; Santa Cruz Biotechnology, sc -376,077), MMA (1:1,000; Cell Signaling Technology, 8015S), ADMA (1:1,000; 13522S; Cell Signaling Technology, 13522S), CARM1 substrate (1:1,000; another gift from Dr. Mark Bedford, MD Anderson Cancer Center, University of Texas), SDMA (1:1,000; Cell Signaling Technology, 13222S), asymmetrically dimethylated SMARCC1 me2a (Arg1064; 1:1,000; Cell Signaling Technology, 94962S), SMARCC1 (1:1,000; Cell Signaling Technology, 11956S), asymmetrically dimethylated PABPC1 (Arg455/Arg460; 1:1,000; Cell Signaling Technology, 3505S), and PABPC1 (1:1,000; Cell Signaling Technology, 4992S) to assess PRMT expression and function.

Techniques: Activity Assay, Expressing, Muscles, Western Blot, Binding Assay, Staining

Journal: Autophagy

Article Title: CARM1 drives mitophagy and autophagy flux during fasting-induced skeletal muscle atrophy.

doi: 10.1080/15548627.2023.2288528

Figure Lengend Snippet: Figure 3. CARM1 regulates metabolic signaling during fasting-induced muscle atrophy. (A) Representative western blots of phosphorylated AMP-activated protein kinase (p-PRKAA/AMPK [Thr172]), total AMPK, PPARGC1A/PGC-1α (PPARG coactivator 1 alpha), CS (citrate synthase), and proteins indicative of mitochondrial OXPHOS (oxidative phosphorylation) complexes I – V (CI – CV) in WT and mKO TA muscles following fasting, accompanied by a typical Ponceau stain. Molecular masses (kDa) are shown at the right of blots. (B-C) graphical summaries of p-PRKAA/AMPK (Thr172), AMPK, AMPK phosphorylation status, PPARGC1A, CS, and total OXPHOS protein expression in WT and mKO TA muscles in response to fasting. Data are expressed as protein content relative to WT fed (n = 8–21). (D) Representative immunofluorescence images of MYH (myosin heavy chain) type I (blue), IIA (green), IIX (red), and IIB (black) in EDL muscles of WT and mKO mice after fasting. (E) graphical summaries of EDL myofiber MYH composition in both genotypes following food deprivation. (F) typical transmission electron micrographs of mitochondria and myonuclei (*) within the SS (subsarcolemmal) and IMF (intermyofibrillar) regions of TA muscle in WT and mKO mice under fed and 48 h fast conditions. Scale bar:

Article Snippet: We employed antibodies against CARM1 (1:5,000; Bethyl Laboratories, A300-421A), PRMT1 (1:1,000; EMD Millipore, 07– 404), PRMT5 (1:1,000; EMD Millipore, 07–405), PRMT6 (1:1,000; Bethyl Laboratories, A300-929A), PRMT7 (1:1,000; Santa Cruz Biotechnology, sc -376,077), MMA (1:1,000; Cell Signaling Technology, 8015S), ADMA (1:1,000; 13522S; Cell Signaling Technology, 13522S), CARM1 substrate (1:1,000; another gift from Dr. Mark Bedford, MD Anderson Cancer Center, University of Texas), SDMA (1:1,000; Cell Signaling Technology, 13222S), asymmetrically dimethylated SMARCC1 me2a (Arg1064; 1:1,000; Cell Signaling Technology, 94962S), SMARCC1 (1:1,000; Cell Signaling Technology, 11956S), asymmetrically dimethylated PABPC1 (Arg455/Arg460; 1:1,000; Cell Signaling Technology, 3505S), and PABPC1 (1:1,000; Cell Signaling Technology, 4992S) to assess PRMT expression and function.

Techniques: Western Blot, Phospho-proteomics, Muscles, Staining, Expressing, Immunofluorescence, Transmission Assay

Journal: Autophagy

Article Title: CARM1 drives mitophagy and autophagy flux during fasting-induced skeletal muscle atrophy.

doi: 10.1080/15548627.2023.2288528

Figure Lengend Snippet: Figure 5. Skeletal muscle autophagy is dysregulated in mKO animals during atrophy. (A) Representative western blots of LC3-I, LC3-II, and SQSTM1 in TA muscles of WT and mKO mice treated with Sal or Col under fed and 48 h fast conditions, accompanied by a typical Ponceau stain. Molecular masses (kDa) are shown at the right of blots. (B-C) Graphical summaries of LC3-II and SQSTM1 flux in WT and mKO animals under fed and 48 h fast conditions. Data are expressed as protein content relative to WT fed (n = 12–16). (D) Representative image from mKO muscle of double-membrane autophagic vacuole (yellow arrow). Scale bar: 200 nm. (E) Graphical summary of total number of autophagic vacuoles in WT and mKO mice treated with Sal or Col under fed and 48 h fast conditions. (F) Heatmap of macroautophagy genes in TA muscle with enrichment unique to carm1 mKO after fasting. Data are expressed relative to WT fed (n = 3). (G) typical western blots of phosphorylated autophagy-related 16 (p-ATG16L1 [Ser278]), total ATG16L1, phosphorylated (p)-MTOR (mechanistic target of rapamycin kinase; Ser2448), total MTOR, p-ULK1 (unc-51 like autophagy activating kinase 1; Ser555), p-ULK1 (Ser757), total ULK1, TFEB (transcription factor EB), SKP2 (S-phase kinase associated protein 2), BECN1, LAMP1

Article Snippet: We employed antibodies against CARM1 (1:5,000; Bethyl Laboratories, A300-421A), PRMT1 (1:1,000; EMD Millipore, 07– 404), PRMT5 (1:1,000; EMD Millipore, 07–405), PRMT6 (1:1,000; Bethyl Laboratories, A300-929A), PRMT7 (1:1,000; Santa Cruz Biotechnology, sc -376,077), MMA (1:1,000; Cell Signaling Technology, 8015S), ADMA (1:1,000; 13522S; Cell Signaling Technology, 13522S), CARM1 substrate (1:1,000; another gift from Dr. Mark Bedford, MD Anderson Cancer Center, University of Texas), SDMA (1:1,000; Cell Signaling Technology, 13222S), asymmetrically dimethylated SMARCC1 me2a (Arg1064; 1:1,000; Cell Signaling Technology, 94962S), SMARCC1 (1:1,000; Cell Signaling Technology, 11956S), asymmetrically dimethylated PABPC1 (Arg455/Arg460; 1:1,000; Cell Signaling Technology, 3505S), and PABPC1 (1:1,000; Cell Signaling Technology, 4992S) to assess PRMT expression and function.

Techniques: Western Blot, Muscles, Staining, Membrane

Journal: Autophagy

Article Title: CARM1 drives mitophagy and autophagy flux during fasting-induced skeletal muscle atrophy.

doi: 10.1080/15548627.2023.2288528

Figure Lengend Snippet: Figure 7. The impact of fasting on human skeletal muscle fiber size, CARM1 biology, and atrophy- and autophagy-related signaling. (A) Representative images of H&E-stained vastus lateralis QUAD muscle cross sections from healthy male participants collected 1 h postprandial (fed) and following 48 h of fasting (fast). Scale bar: 50 μm. (B) graphical summary of the average myofiber CSA of QUAD muscles from adult humans in response to fasting. Data are expressed as CSA relative to fed (n = 9). (C) distribution of CSA (μm2) in QUAD of fed (solid line) and fast (dashed line) human myofibers (n = 9). (D) heatmap visualizing the average mRNA expression of PRMT family members in QUAD muscle of adult humans during fed and 40 h fast conditions. Microarray data extracted from GSE28016 (n = 7). (E) Representative western blots of CARM1, asymmetric arginine dimethylated CARM1 substrates, SMARCC1 me2a (Arg1064), total SMARCC1, PABPC1 me2a (Arg455/Arg460), total PABPC1, PRKN, BNIP3, LC3-I, LC3-II, p-ATG16L1 (Ser278), total ATG16L1, p-ULK1 (Ser757), and total ULK1 in QUAD muscles from healthy humans in response to 48 h of fasting, accompanied by a typical Ponceau stain. Molecular masses (kDa) are shown at the right of blots. (F-N) graphical summaries of CARM1, CARM1 substrate,

Article Snippet: We employed antibodies against CARM1 (1:5,000; Bethyl Laboratories, A300-421A), PRMT1 (1:1,000; EMD Millipore, 07– 404), PRMT5 (1:1,000; EMD Millipore, 07–405), PRMT6 (1:1,000; Bethyl Laboratories, A300-929A), PRMT7 (1:1,000; Santa Cruz Biotechnology, sc -376,077), MMA (1:1,000; Cell Signaling Technology, 8015S), ADMA (1:1,000; 13522S; Cell Signaling Technology, 13522S), CARM1 substrate (1:1,000; another gift from Dr. Mark Bedford, MD Anderson Cancer Center, University of Texas), SDMA (1:1,000; Cell Signaling Technology, 13222S), asymmetrically dimethylated SMARCC1 me2a (Arg1064; 1:1,000; Cell Signaling Technology, 94962S), SMARCC1 (1:1,000; Cell Signaling Technology, 11956S), asymmetrically dimethylated PABPC1 (Arg455/Arg460; 1:1,000; Cell Signaling Technology, 3505S), and PABPC1 (1:1,000; Cell Signaling Technology, 4992S) to assess PRMT expression and function.

Techniques: Staining, Muscles, Expressing, Microarray, Western Blot