rabbit polyclonal anti brf1 2 zfp36l1 2  (Cell Signaling Technology Inc)

Journal: PLoS ONE

Article Title: ZFP36L1 Negatively Regulates Plasmacytoid Differentiation of BCL1 Cells by Targeting BLIMP1 mRNA

doi: 10.1371/journal.pone.0052187

Figure Lengend Snippet: (A) RT-PCR analysis of ZFP36L1 expression in human malignant B cells representing different stages of B cell differentiation. For comparison BLIMP1 expression was also measured. β ACTIN expression levels are shown as a loading control. (B) Comparison of ZFP36L1 protein levels in human Ramos B cells before and after 3 h PMA stimulation and in two myeloma cell lines RPMI-8226 cells and KMS-11. BLIMP1 expression is also shown in RPMI-8226 cells and KMS-11 cells. HSP90 levels are shown as a loading control. ZFP36L1/L2, HSP90 and BLIMP1 proteins were detected by anti-BRF1/2, anti-HSP90 and anti-BLIMP1 antibodies respectively. Anti-BRF1/2 antibody cross-reacts with ZFP36L2 (approx. 60 kDa) and ZFP36L1 appears typically as a constellation of induced bands (approx. 40 kDa) in human cells.

Article Snippet: Nitrocellulose membranes were incubated with appropriate dilution of the following primary antibodies for 1h; rabbit polyclonal anti-BRF1/2 (ZFP36L1/2) (Cell Signalling, Danvers, MA, USA), goat anti-ZFP36 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BLIMP1 (Cell Signalling).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Cell Differentiation

Journal: PLoS ONE

Article Title: ZFP36L1 Negatively Regulates Plasmacytoid Differentiation of BCL1 Cells by Targeting BLIMP1 mRNA

doi: 10.1371/journal.pone.0052187

Figure Lengend Snippet: downregulation of ZFP36L1 is associated with plasmacytoid differentiation of B cells. (A) Western Blot analysis of ZFP36L1 expression in IL-2/5 treated murine leukemic BCL1 cells. Protein lysates were made from unstimulated cells (lane 1) or from cells 96 h after stimulation with cytokines (20 ng/ml IL-2 and 5 ng/ml IL-5) (lane 2). ZFP36L1 and HSP90 proteins were detected by anti-BRF1/2 and anti-HSP 90 antibodies respectively. (B) qRT-PCR analysis of zfp36l1 and blimp1 mRNA expression in day 0 versus 48 h IL-2/5 stimulated BCL1 cells. (C) qRT-PCR analysis of time course of zfp36l1 and blimp1 mRNA expression over 3 days in LPS (10 µg/ml) stimulated murine splenic B cells. The 2 –ΔΔCT method of relative quantification was used to determine the fold change in mRNA expression. The results shown were normalized to β-actin mRNA expression. The results show mean ±SD from one representative experiment.

Article Snippet: Nitrocellulose membranes were incubated with appropriate dilution of the following primary antibodies for 1h; rabbit polyclonal anti-BRF1/2 (ZFP36L1/2) (Cell Signalling, Danvers, MA, USA), goat anti-ZFP36 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BLIMP1 (Cell Signalling).

Techniques: Western Blot, Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: ZFP36L1 Negatively Regulates Plasmacytoid Differentiation of BCL1 Cells by Targeting BLIMP1 mRNA

doi: 10.1371/journal.pone.0052187

Figure Lengend Snippet: IgM production. Levels of IgM production in ZFP36L1 transfected BCL1 stimulated with IL-2/5 (20 ng/ml IL-2 and 5 ng/ml IL-5) over 4 days compared to levels produced by empty vector BCL1 cells cultured under the same conditions. Cells were co-transfected with either pcDNA3.ZFP36L1 or empty pcDNA3 vector and pcDNA3.EGFP and then sorted on the basis of EGFP expression before been set up in culture in the absence or presence of cytokines. Cells were cultured at 4.0×10 4 /ml and ELISA measurements were made in duplicate. The results shown are representative of 3 similar experiments.

Article Snippet: Nitrocellulose membranes were incubated with appropriate dilution of the following primary antibodies for 1h; rabbit polyclonal anti-BRF1/2 (ZFP36L1/2) (Cell Signalling, Danvers, MA, USA), goat anti-ZFP36 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BLIMP1 (Cell Signalling).

Techniques: Transfection, Produced, Plasmid Preparation, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: ZFP36L1 Negatively Regulates Plasmacytoid Differentiation of BCL1 Cells by Targeting BLIMP1 mRNA

doi: 10.1371/journal.pone.0052187

Figure Lengend Snippet: (A) qRT-PCR analysis of zfp36l1 mRNA levels in pSicoR.zfp36l1.RNAi1 and pSicoR.zfp36l1.RNAi2 lentivirus infected cells compared to wild-type, empty vector or pSicoR.scramble.RNAi infected cells. The results represent mean ±SD (n = 3) of zfp36l1 mRNA levels in three independent cells lines generated by three independent rounds of lentiviral infection for each cell type (apart from wild-type). * = p<0.05 as determined by t-test. (B) Western blot analysis of ZFP36L1 protein expression in wild-type, empty vector, scramble, pSicoR.scramble.RNAi1and pSicoR.scramble.RNAi2 cells. HSP90 levels are shown as a loading control.

Article Snippet: Nitrocellulose membranes were incubated with appropriate dilution of the following primary antibodies for 1h; rabbit polyclonal anti-BRF1/2 (ZFP36L1/2) (Cell Signalling, Danvers, MA, USA), goat anti-ZFP36 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BLIMP1 (Cell Signalling).

Techniques: Quantitative RT-PCR, Infection, Plasmid Preparation, Generated, Western Blot, Expressing

Journal: PLoS ONE

Article Title: ZFP36L1 Negatively Regulates Plasmacytoid Differentiation of BCL1 Cells by Targeting BLIMP1 mRNA

doi: 10.1371/journal.pone.0052187

Figure Lengend Snippet: Cell numbers in the absence (A) and presence (B) of cytokines (IL-2 and IL-5) in pSicoR.zfp36l1.RNAi1 and pSicoR.zfp36l1.RNAi2 BCL1 cells compared to compared to wild-type, empty vector or pSicoR.scramble.RNAi infected cells. Cells numbers were counted in quadruplicate 4 days after seeding 2×10 5 /ml cells on day 0 in flasks in the absence or presence of cytokines (IL-2 20 ng/ml and IL-5 5 ng/ml). Mean ±SD (n = 3) are shown for three independent cell lines generated for each of the lentivirus infected cells. IgM secretion (ng/ml) measured by ELISA, 4 days after the start of the culture, in the absence (C) and presence (D) of cytokines (IL-2 20 ng/ml and IL-5 5 ng/ml) in pSicoR.zfp36l1.RNAi1 and pSicoR.zfp36l1.RNAi2 BCL1 cells compared to compared to wild-type, empty vector or pSicoR.scramble.RNAi infected cells. Mean ±SD (n = 3) are shown for three independent cell lines generated for each of the lentivirus infected cells. ** = p<0.01, * = p<0.05 as determined by t-test.

Article Snippet: Nitrocellulose membranes were incubated with appropriate dilution of the following primary antibodies for 1h; rabbit polyclonal anti-BRF1/2 (ZFP36L1/2) (Cell Signalling, Danvers, MA, USA), goat anti-ZFP36 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BLIMP1 (Cell Signalling).

Techniques: Plasmid Preparation, Infection, Generated, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: ZFP36L1 Negatively Regulates Plasmacytoid Differentiation of BCL1 Cells by Targeting BLIMP1 mRNA

doi: 10.1371/journal.pone.0052187

Figure Lengend Snippet: (A) Heat map expression profile of known ZFP36L1 target genes in terminal B cell differentiation. The profiles are compared with the reference genes, ZFP36L1, BLIMP1 and XBP1 (first three rows). GMCSF, VEGFA and IL-3 are validated ZFP36L1 targets; the remaining genes have been validated for ZFP36 . Data was taken from GEO Accession number GSE 6691 . Red indicates high and blue, low expression. Key: CLL: B chronic lymphocytic leukemia, MM: multiple myeloma, WM-BL: Waldenstrom’s macroglobulinemia B cells, WM-PB: Waldenstrom’s macroglobulinemia plasma cells, NBC: normal B cells, PC: normal plasma cells. (B) Graphical representation of the ARACNe network for BLIMP1 and ZFP36L1. Nodes representing the BLIMP1 and ZFP36L1 hubs are shown enlarged. Only first neighbours of these hubs are shown in the module. Nodes representing inferred BLIMP1 targets (significantly up-regulated in normal B cells) are blue; nodes representing inferred ZFP36L1 targets (significantly up-regulated in normal plasma cells) are red. Network graphics were generated as group attribute layout using Cytoscape version 2.6.0. .

Article Snippet: Nitrocellulose membranes were incubated with appropriate dilution of the following primary antibodies for 1h; rabbit polyclonal anti-BRF1/2 (ZFP36L1/2) (Cell Signalling, Danvers, MA, USA), goat anti-ZFP36 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BLIMP1 (Cell Signalling).

Techniques: Expressing, Cell Differentiation, Generated

Journal: PLoS ONE

Article Title: ZFP36L1 Negatively Regulates Plasmacytoid Differentiation of BCL1 Cells by Targeting BLIMP1 mRNA

doi: 10.1371/journal.pone.0052187

Figure Lengend Snippet: qRT-PCR analysis of blimp1, xbp1, irf4 and bcl6 mRNA expression in pSicoR.zfp36l1.RNAi1 and pSicoR.zfp36l1.RNAi2 BCL1 cells compared to compared to wild-type, empty vector or pSicoR.scramble.RNAi infected cells. Cells were cultured in medium alone for 48h. Total RNA was extracted from 5×10 6 cells, 1 µg RNA was reverse transcribed and the resulting cDNA was used as template for qRT-PCR assay with mouse gene specific primers for blimp1, xbp1, irf4 and bcl6. The 2 –ΔΔCT method of relative quantification was used to determine the fold change in mRNA expression compared to levels in wild-type cells. Mean ±SD (n = 3) are shown for three independent cell lines generated for each of the lentivirus infected cells. * = p<0.05 as determined by t-test.

Article Snippet: Nitrocellulose membranes were incubated with appropriate dilution of the following primary antibodies for 1h; rabbit polyclonal anti-BRF1/2 (ZFP36L1/2) (Cell Signalling, Danvers, MA, USA), goat anti-ZFP36 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BLIMP1 (Cell Signalling).

Techniques: Quantitative RT-PCR, Expressing, Plasmid Preparation, Infection, Cell Culture, Generated

Journal: PLoS ONE

Article Title: ZFP36L1 Negatively Regulates Plasmacytoid Differentiation of BCL1 Cells by Targeting BLIMP1 mRNA

doi: 10.1371/journal.pone.0052187

Figure Lengend Snippet: (A) Western blot analysis of BLIMP1 levels in control and ZFP36L1 knockdown cells. BLIMP1 expression levels are upregulated in ZFP36L1 knockdown cells compared to controls. βACTIN levels are shown as a loading control. (B) ZFP36L1 mediates degradation of the BLIMP1 3′UTR. HEK 293 T cells were transfected with pMIRBLIMP1 3′UTR construct alone (control) or with either ZFP36L1 or a zinc finger domain mutant, ZFP36L1 Mut. Renilla luciferase was also included in all transfections as a normalization control. 24 hours later cell lysates were harvested and firefly and renilla luciferase levels measured using a Fluorstar Optima plate reader. Relative levels of luciferase activity were measured. Mean ±SD, are shown for four independent experiments (n = 4), * = p<0.05 as determined by t-test.

Article Snippet: Nitrocellulose membranes were incubated with appropriate dilution of the following primary antibodies for 1h; rabbit polyclonal anti-BRF1/2 (ZFP36L1/2) (Cell Signalling, Danvers, MA, USA), goat anti-ZFP36 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BLIMP1 (Cell Signalling).

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