Journal: eLife

Article Title: Overexpression screen of interferon-stimulated genes identifies RARRES3 as a restrictor of Toxoplasma gondii infection

doi: 10.7554/eLife.73137

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-IRF1 D5E4 (rabbit monoclonal) , Cell Signaling Technology , Cat#: 8478S; RRID: AB_10949108 , (1:500).

Techniques: Expressing, Luciferase, Staining, Transfection, Recombinant, Plasmid Preparation, Virus, CyQUANT Assay, LDH Cytotoxicity Assay, Cell Culture, Lysis, Amplification, DNA Amplification, Software

Journal: Journal of Cellular and Molecular Medicine

Article Title: RNF 168 facilitates oestrogen receptor ɑ transcription and drives breast cancer proliferation

doi: 10.1111/jcmm.13694

Figure Lengend Snippet: Reduction of RNF 168 level reduces recruitment of RNF 168 to ER ɑ promoter—a potential mechanism for ER ɑ signalling regulation. A, Genomic organization of ER α promoter structure of human ER α genes is shown, among which promoter A, promoter B and promoter E2 are used in MCF ‐7 cells. B, Ch IP assay shows that RNF 168 is recruited to ER α promoter B and E2. MCF 7 cells were fixed for 30 min. Rabbit Ig G was used as the negative control, while ER α antibody was used as the positive control. The primer sequences were shown in Table S1. Then enriched DNA fragments were subject to PCR reaction and detected by DNA gel electrophoresis. C, Ch IP assay shows that for RNF 168 depletion decreases RNF 168 recruitment to ER α promoter regions. MCF 7 cells were transfected with si RNF 168 or siControl for 48 h. After that, cells were fixed for 30 min. Rabbit Ig G was used as the negative control. The primer sequences were shown in Table S1. The relative ER α promoter enrichment was measured by real‐time PCR . * P < .05; ** P < .01; *** P < .001 for binding comparison. D, Ch IP assay shows that RNF 168, ER α, ER α co‐activators ( SRC 1 and SRC 3) and H3K27ac co‐occupy at ER α promoter B and E2. MCF 7 cells were fixed for 30 min. Rabbit Ig G was used as the negative control. The primer sequences were shown in Table S1. Then enriched DNA fragments were subject to PCR reaction and detected by DNA gel electrophoresis. E, Ch IP assay shows that for RNF 168 depletion decreases ER α, ER α co‐activators ( SRC 1 and SRC 3) and H3K27ac recruitment to ER α promoter regions. MCF 7 cells were transfected with si RNF 168 or siControl for 48 h. After that, cells were fixed for 30 min. Rabbit Ig G was used as the negative control. The primer sequences were shown in Table S1. The relative ER α promoter enrichment was measure by real‐time PCR . * P < .05; ** P < .01; *** P < .001 for binding comparison

Article Snippet: Anti‐SRC1 (128E7), anti‐SRC3 (5E11) and anti‐H3K27ac (D5E4) antibodies were acquired from Cell Signaling Technology.

Techniques: Negative Control, Positive Control, DNA Gel Electrophoresis, Transfection, Real-time Polymerase Chain Reaction, Binding Assay

Journal: Metabolites

Article Title: TCF19 Impacts a Network of Inflammatory and DNA Damage Response Genes in the Pancreatic β-Cell

doi: 10.3390/metabo11080513

Figure Lengend Snippet: MAGIC analysis on the list of upregulated genes after TCF19 expression in INS-1 cells identifies significant enrichment for genes with known ChIP signals for STAT1, STAT2, and IRF1 in their promoters (FDR < 10%). A higher score reflects increased likelihood that the factor is enriched for binding gene regions in the list of upregulated genes. Genes included in analysis were those that were upregulated more than 2-fold with an associated FDR < 5%.

Article Snippet: Primary antibodies and dilutions were as follows: Myc antibody (9E10:sc-40, Santa Cruz Biotechnology, 1:1000), Beta actin (8H10D10, Cell Signaling, 1:1000), STAT1 (#9172, Cell Signaling, 1:1000), phospho- STAT1 Ser727 (#9177, Cell Signaling, 1:1000), phospho-STAT1 Tyr701 (D4A7 #7649, Cell Signaling, 1:1000), phospho- NF-κB p65 Ser536 (93H1 #3033, Cell Signaling, 1:1000), NF-κB p65 (D14E12 XP #8242, Cell Signaling, 1:1000), IRF1 (D5E4, Cell Signaling, 1:1000), GAPDH (14C10, Cell Signaling, 1:1000), Beta-tubulin (#2146, Cell Signaling, 1:1000) in 5% BSA-TBST or 5% non-fat milk.

Techniques: Expressing, Binding Assay

Journal: Metabolites

Article Title: TCF19 Impacts a Network of Inflammatory and DNA Damage Response Genes in the Pancreatic β-Cell

doi: 10.3390/metabo11080513

Figure Lengend Snippet: Overexpression of TCF19 in INS-1 cells does not lead to increased activation of transcription factor targets but leads to decreased activation of STAT1 Y701 phosphorylation. Representative images of two western blot replicates along with analysis of all replicates are shown. Densitometry with Image J 1.44o was used to quantify the bands on the western blots, which were then normalized to the housekeeper protein band. ( A ) Serine 727 phospho-STAT1/STAT1 protein expression does not show a statistically significant difference between control and hsTCF19 overexpressing cells (n = 5) (phospho-STAT1~91 kDa, STAT1~84, 91 kDa). ( B ) Tyrosine 701 phospho-STAT1/STAT1 protein expression shows statistically significant decrease in hsTCF19 overexpressing cells compared to control (n = 3) (phospho-STAT1~84, 91 kDa). ( C ) IRF1 protein levels are not significantly different. Representative western blots in the figure have IRF1 levels normalized to GAPDH. Three other replicates are normalized to beta tubulin (n = 5) (IRF1~48 kDa). ( D ) There is no difference in phospho NF-κB/NF-κB levels with hsTCF19 overexpression (n = 3) (phospho NF-κB~65 kDa, NF-κB~65 kDa). All data are means ± SEM * p < 0.05.

Article Snippet: Primary antibodies and dilutions were as follows: Myc antibody (9E10:sc-40, Santa Cruz Biotechnology, 1:1000), Beta actin (8H10D10, Cell Signaling, 1:1000), STAT1 (#9172, Cell Signaling, 1:1000), phospho- STAT1 Ser727 (#9177, Cell Signaling, 1:1000), phospho-STAT1 Tyr701 (D4A7 #7649, Cell Signaling, 1:1000), phospho- NF-κB p65 Ser536 (93H1 #3033, Cell Signaling, 1:1000), NF-κB p65 (D14E12 XP #8242, Cell Signaling, 1:1000), IRF1 (D5E4, Cell Signaling, 1:1000), GAPDH (14C10, Cell Signaling, 1:1000), Beta-tubulin (#2146, Cell Signaling, 1:1000) in 5% BSA-TBST or 5% non-fat milk.

Techniques: Over Expression, Activation Assay, Phospho-proteomics, Western Blot, Expressing, Control

Journal: Journal of Neurosurgery

Article Title: Immunohistochemical analysis of histone H3 acetylation in the trigeminal root entry zone in an animal model of trigeminal neuralgia

doi: 10.3171/2018.5.jns172948

Figure Lengend Snippet: FIG. 6. Immunofluorescence staining of histone H3K27 acetylation in the CNS-PNS transitional zone of the TREZ. The level of histone H3K27 acetylation cells (green) in the sham group (A–D) was relatively lower than that in the TN group after operation. In the TN group (E–H), the amount of immunoreactive acetylated H3K27 cells (green) decreased from postoperative day 7 to day 28. Merged images show the positive ratio of immunoreactive acetylated H3K27 cells (green) among the DAPI-positive cells (blue) around the CNS-PNS transitional zone of the TREZ (same area as in the white dashed box in Fig. 3A) in the sham group (A1–D1) and the TN group (E1–H1). The ratio of H3K27 acetylated cells in the TN group was higher than that in the sham operation group (I). Asterisks indicate a significant difference (p < 0.05). Oligodendrocytes (arrows, J), astrocytes (triangles), and Schwann cells (double arrows, K) were acetylated in the TREZ. Bar = 150 μm (A–H1) and 50 μm (J and K). Figure is available in color online only.

Article Snippet: Methods Antibodies Acetyl histone H3K9 (C5B11) rabbit monoclonal antibody (mAb; 1:50 dilution), acetyl histone H3K18 (D8Z5H) rabbit mAb (1:50 dilution), and acetyl histone H3K27 (D5E4) rabbit mAb (1:50 dilution) primary antibodies were purchased from Cell Signaling Technology.

Techniques: Immunofluorescence, Staining