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

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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: Nucleic Acids Research

Article Title: The protein arginine methyltransferases CARM1 and PRMT1 cooperate in gene regulation

doi: 10.1093/nar/gkn166

Figure Lengend Snippet: A subset of downregulated microarray targets is inducible by IL-4 treatment depending on the presence of STAT5, CARM1 and PRMT1. ( A ) HeLa cells were treated without (0 h) or with IL-4 for the indicated times (1 h and 4 h). Subsequently, cells were harvested in SDS-lysis buffer and 50 μl of each sample were analysed by Western Blot with the indicated antibodies. ( B ) HeLa cells were treated without (−) or with (+) IL-4 for 16 h. Subsequently, cells were harvested and total RNA was analysed by RT–QPCR for transcript levels of CITED2 and KRT8 normalized for GAPDH. ( C ) HeLa cells were transfected with siNON-targeting, siCARM1/siPRMT1, siSTAT5 or siSTAT6 for 24 h and harvested in SDS-lysis buffer. Fifty microlitres of each sample were analysed by Western Blot with the indicated antibodies. ( D ) HeLa cells were transfected with siNON-targeting, siCARM1/siPRMT1, siSTAT5 or siSTAT6 for 24 h. Subsequently, cells were treated without (−, black bars) or with (+, grey bars) IL-4 for 24 h. Total RNA was analysed by RT–QPCR for transcript levels of CARM1, PRMT1 and CITED2 normalized for GAPDH.

Article Snippet: The following antibodies were employed: anti-PRMT1 from Upstate (07-404), anti-PRMT1 generated in rabbits against recombinant human PRMT1 protein (aa 1–343), anti-CARM1 from Upstate (07-080), anti-CARM1 generated in rabbits against recombinant murine PRMT4 protein (aa 433–608), anti-H4 R3me2 from Upstate (07-213), anti-H3 R17me2 from Abcam (ab8284), anti-STAT5a/b from Santa Cruz, CA, USA (sc-835), anti-phospho Tyr694/699 STAT5 from Cell Signaling (9351), anti-STAT6 from Santa Cruz (sc-981), anti-phospho Tyr641 STAT6 from Cell Signaling (9361) and anti-β tubulin (MAB3408) from Chemicon, Billerica, MA, USA.

Techniques: Microarray, Lysis, Western Blot, Quantitative RT-PCR, Transfection

Journal: Nucleic Acids Research

Article Title: The protein arginine methyltransferases CARM1 and PRMT1 cooperate in gene regulation

doi: 10.1093/nar/gkn166

Figure Lengend Snippet: CARM1 and PRMT1 are recruited in an IL-4-dependent manner to the endogenous CITED2 gene promoter and cooperatively coactivate STAT5 in an IL-4-dependent manner. ( A ) Starved HeLa cells (0.1% FCS) were treated without (−) or with (+) IL-4 and/or 10% FCS for 16 h. Subsequently, cells were harvested and total RNA was analysed by RT–QPCR for transcript levels of CITED2 normalized for GAPDH. ( B ), Scheme of the CITED2 gene illustrating the location of the putative STAT5-binding site within the promoter. ( C and D ) Starved HeLa cells (0.1% FCS) were treated without (−, black bars) or with 10% FCS/IL-4 (+, grey bars) for 16 h. Subsequently, cells were harvested and subjected to ChIP analysis using antibodies against STAT5 (corresponding control antibody: IgG), PRMT1 (corresponding control antibody: preimmune-PRMT1) and CARM1 (corresponding control antibody: preimmune-CARM1). Immunoprecipitated DNA was analysed in triplicates by QPCR with primers for the CITED2 gene promoter encompassing the STAT5-binding site (in C) and an intergenic control region 6-kb upstream of the CITED gene (in D). Mean values were expressed as fold control IgG, which was equated 1. ( E ) Starved HeLa cells (0.1% FCS) were treated without (−, black bars) or with 10% FCS/IL-4 for 2, 8 and 16 h. Subsequently, cells were harvested and subjected to ChIP analysis using control antibodies (IgG), antibodies against STAT5, H4 R3me2, H3 R17me2, PRMT1 and CARM1. Immunoprecipitated DNA was analysed in triplicates by QPCR with primers for the CITED2 gene promoter as in C. ( F ) HeLa cells were transiently transfected with a (STAT5-RE) 6 -luciferase (luc) reporter gene constructs and a CMV-β-galactosidase reporter (the latter for normalization) in the absence or presence of expression constructs for STAT5b, CARM1 and PRMT1. Twenty-four hours after transfection medium was changed and cells were treated without (−, black bars) or for 16 h with IL-4 (+, grey bars). Subsequently, cells were harvested and assayed for luciferase activity. Values given are the mean of triplicate measurements and expressed relative to the β-galactosidase activity. Equal protein expression was confirmed by Western Blot (data not shown).

Article Snippet: The following antibodies were employed: anti-PRMT1 from Upstate (07-404), anti-PRMT1 generated in rabbits against recombinant human PRMT1 protein (aa 1–343), anti-CARM1 from Upstate (07-080), anti-CARM1 generated in rabbits against recombinant murine PRMT4 protein (aa 433–608), anti-H4 R3me2 from Upstate (07-213), anti-H3 R17me2 from Abcam (ab8284), anti-STAT5a/b from Santa Cruz, CA, USA (sc-835), anti-phospho Tyr694/699 STAT5 from Cell Signaling (9351), anti-STAT6 from Santa Cruz (sc-981), anti-phospho Tyr641 STAT6 from Cell Signaling (9361) and anti-β tubulin (MAB3408) from Chemicon, Billerica, MA, USA.

Techniques: Quantitative RT-PCR, Binding Assay, Control, Immunoprecipitation, Transfection, Luciferase, Construct, Expressing, Activity Assay, Western Blot

Journal: Nucleic Acids Research

Article Title: The protein arginine methyltransferases CARM1 and PRMT1 cooperate in gene regulation

doi: 10.1093/nar/gkn166

Figure Lengend Snippet: CARM1 and PRMT1 interaction with STAT5 is enhanced by IL-4. ( A ) GST alone (control), GST-CARM1 and GST-PRMT1 were expressed in E. coli and purified. Equal amounts of these GST-fusions, as indicated in the Coomassie-stained SDS-gel (bottom panel, full-length protein bands marked with asterisks), coupled to glutathione beads were used in pulldown experiments in the presence of HeLa whole-cell extract overexpressing STAT5b and treated without or with IL-4 for 16 h. Interactions between the proteins were detected by anti-STAT5 Western Blot (top panel). One percent input of HeLa whole-cell extract is shown. ( B ) HeLa cells were left unstimulated or stimulated with IL-4 for 1 or 16 h. Subsequently, nuclear extracts were prepared and subjected to immunoprecipitation with antibodies against PRMT1, CARM1 and as control IgG. Immunoprecipitates were resolved by SDS–PAGE and analysed by Western Blot analysis with the indicated antibodies to confirm the immunoprecipitation of PRMT1 and CARM1 (as control) and to investigate STAT5 coimmunoprecipitation. Ten percent input (50 μg) of HeLa cell extract is shown.

Article Snippet: The following antibodies were employed: anti-PRMT1 from Upstate (07-404), anti-PRMT1 generated in rabbits against recombinant human PRMT1 protein (aa 1–343), anti-CARM1 from Upstate (07-080), anti-CARM1 generated in rabbits against recombinant murine PRMT4 protein (aa 433–608), anti-H4 R3me2 from Upstate (07-213), anti-H3 R17me2 from Abcam (ab8284), anti-STAT5a/b from Santa Cruz, CA, USA (sc-835), anti-phospho Tyr694/699 STAT5 from Cell Signaling (9351), anti-STAT6 from Santa Cruz (sc-981), anti-phospho Tyr641 STAT6 from Cell Signaling (9361) and anti-β tubulin (MAB3408) from Chemicon, Billerica, MA, USA.

Techniques: Control, Purification, Staining, SDS-Gel, Western Blot, Immunoprecipitation, SDS Page