affinity purified wb  (Bethyl)

Journal: Molecular Cell

Article Title: TFIIIC Binding to Alu Elements Controls Gene Expression via Chromatin Looping and Histone Acetylation

doi: 10.1016/j.molcel.2019.10.020

Figure Lengend Snippet:

Article Snippet: rabbit anti-GTF3C1/TFIIIC220 Antibody, Affinity Purified WB , Bethyl , RRID: AB_938042.

Techniques: Affinity Purification, Virus, Recombinant, Protease Inhibitor, Sequencing, Multiplex Assay, Bicinchoninic Acid Protein Assay, cDNA Synthesis, SYBR Green Assay, Electrophoresis, Gene Expression, Expressing, Plasmid Preparation, Software

Journal: International immunology

Article Title: Inhibition of lipopolysaccharide-mediated NFkappaB activation by ethanol in human monocytes.

doi: 10.1093/intimm/11.11.1781

Figure Lengend Snippet: Fig. 2. Ethanol inhibits nuclear NFκB binding in peripheral blood monocytes. Adherence-isolated monocytes were stimulated with 1 µg/ml LPS in the presence or absence of 25 mM ethanol for 1 h. Nuclear extracts were then prepared and gel shift analysis performed as described in Methods. (A) Representative EMSA of three separate experiments done using [32P]NFκB consensus oligonucleotide. A 20-fold excess of the unlabeled NFκB fragment was included as a cold competitor (comp). The extent of DNA binding was quantitated using the NIH Image program and densitometric units are indicated at the bottom of each lane. (B) EMSA showing better resolution of the NFκB complexes, achieved by using 0.253TBE buffer in native gel electrophoresis. Corresponding densitometric units of the individual complexes are shown at the bottom of each lane. Complex I, p65/p50 heterodimer; Complex II, p50/p50 homodimer.

Article Snippet: NFκB p50 (SC114) affinity-purified rabbit polyclonal antibody was obtained from Santa Cruz Biotechnology (P , 0.001, n 5 15).

Techniques: Binding Assay, Isolation, Gel Shift, Nucleic Acid Electrophoresis

Journal: International immunology

Article Title: Inhibition of lipopolysaccharide-mediated NFkappaB activation by ethanol in human monocytes.

doi: 10.1093/intimm/11.11.1781

Figure Lengend Snippet: Fig. 3. Supershift analysis of NFκB in ethanol-treated monocytes. Nuclear extracts from monocytes stimulated for 1 h with 1 µg/ml LPS with or without 25 mM ethanol were subjected to gel shift analysis. All reactions were admixed with rabbit anti-p50 antibody (2 µg) (Santa Cruz Biotechnology) (A) or with anti-human-p65 antisera (generously provided by Dr Nancy Rice) (B) after addition of [32P]NFκB oligonucleotide and incubated for 30 min. Shifted bands were detected by their retarded mobility.

Article Snippet: NFκB p50 (SC114) affinity-purified rabbit polyclonal antibody was obtained from Santa Cruz Biotechnology (P , 0.001, n 5 15).

Techniques: Gel Shift, Incubation

Journal: International immunology

Article Title: Inhibition of lipopolysaccharide-mediated NFkappaB activation by ethanol in human monocytes.

doi: 10.1093/intimm/11.11.1781

Figure Lengend Snippet: Fig. 4. Lack of translocation of NFκB subunits in ethanol-treated monocytes. Monocytes were stimulated with 1 µg/ml LPS in the presence or absence of 25 mM ethanol for 1 h. Nuclear and cytoplasmic extracts (10 µg/group) were subjected to immunoblotting using anti-p50 antibody (1:500 dilution) (A) or anti-p65 antibody (1:1000 dilution) (B) and visualized by ECL assay (Amersham). Results are representative of four experiments with different blood donors.

Article Snippet: NFκB p50 (SC114) affinity-purified rabbit polyclonal antibody was obtained from Santa Cruz Biotechnology (P , 0.001, n 5 15).

Techniques: Translocation Assay, Western Blot

Journal: International immunology

Article Title: Inhibition of lipopolysaccharide-mediated NFkappaB activation by ethanol in human monocytes.

doi: 10.1093/intimm/11.11.1781

Figure Lengend Snippet: Fig. 5. Ethanol decreases LPS-induced phospho- IκBα levels without Fig. 6. Protein synthesis is required for ethanol-mediated inhibition affecting total cytoplasmic IκBα. Cytoplasmic extracts (20 µg/group) of LPS-induced NFκB activation. Adherence-isolated monocytes were of monocytes stimulated with 1 µg/ml LPS with or without 25 mM treated with 10 µg/ml cycloheximide (cyclo) for 30 min before the ethanol for 1 h were subjected to immunoblotting using the anti-IκBα addition of 1 µg/ml LPS in the presence or absence of 25 mM ethanol antibody (Santa Cruz Biotechnology) (A) or phospho-specific anti- for 1 h. (A) Cytoplasmic extracts were subjected to Western blot IκBα (Ser32) antibody (B) as described in Methods. Densitometric analysis using the anti-IκBα antibody. (B) NFκB activation in the values for the individual bands are indicated below each lane. nuclear extracts of the same samples was determined by the gel shift analysis. Data are representative of four experiments showing similar results. provides a mechanism to terminate ongoing NFκB activation (25,26). Thus, one can hypothesize that ethanol may affect the p65/p50 heterodimer translocation and DNA binding protein synthesis is not required for LPS-mediated NFκB activation (27), there was no change in LPS-induced NFκB activity by increasing the re-accumulation of the IκBα pool in the cytosol. To check whether ethanol has any effect on the in cycloheximide pre-treated monocytes (Fig. 6B). These results suggest that de novo protein synthesis is required for re-synthesis of IκBα protein, we performed experiments using the protein synthesis inhibitor cycloheximide (10 µg/ml) in ethanol-induced inhibition of NFκB activation. Thus, these data together suggest that ethanol-induced inhibition of LPS- combination with all the stimulations used in the previous experiments. Data in Fig. 6(A) demonstrate that substantial induced NFκB is a complex phenomenon and may require an association of synthetic cellular responses in human IκBα levels were detected in unstimulated monocytes. Cyclo- heximide partially inhibited the recovery of IκBα levels in LPS- monocytes. stimulated monocytes at 1 h, suggesting that accumulation of IκBα in monocytes after activation requires de novo protein Discussion synthesis. Monocytes pretreated with cycloheximide for 30 min and then with LPS 1 ethanol showed a substantial The goal of this study was to define the effect of acute alcohol treatment on LPS-induced NFκB activation and to characterize reduction in IκBα compared to the LPS 1 ethanol-stimulated (no cycloheximide) cells. This indicates that ethanol affects the molecular mechanisms involved in the ethanol-mediated inhibition of LPS-induced pro-inflammatory cytokines. Here, IκBα by a mechanism that requires de novo protein synthesis. Enhanced IκBα re-synthesis, which in turn may shut off LPS- we show for the first time that ethanol affects LPS-induced NFκB activation in human monocytes by down-regulation of induced NFκB (p65/p50) activation and nuclear translocation, is a likely mechanism for ethanol’s effect. However, increased its DNA binding activity. Our results also demonstrate that the LPS-induced translocation of the p65/50 heterodimer is cytoplasmic IκBα would be expected in such cases. Finally, we tested whether de novo protein synthesis was prevented by ethanol treatment in human monocytes. One of the significant points of this study is that since the nuclear required for inhibition of LPS-induced NFκB nuclear binding by ethanol. We found that cycloheximide prevented the ethanol- migration and DNA binding of NFκB has been previously associated with LPS-mediated transcriptional activation of the mediated inhibition of NFκB activation in LPS-stimulated monocytes. Consistent with previous studies showing that TNF-α and IL-1 gene in primary macrophages, alcohol may

Article Snippet: NFκB p50 (SC114) affinity-purified rabbit polyclonal antibody was obtained from Santa Cruz Biotechnology (P , 0.001, n 5 15).

Techniques: Inhibition, Activation Assay, Isolation, Western Blot, Gel Shift, Translocation Assay, Binding Assay, Activity Assay, Migration