CREB [p Ser133] Antibody NB300-273 from Novus Biologicals, a Bio-Techne Brand

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RNA biology
October 15 2021

Fig 1: Short-term (1 hr) forskolin treatment increases the expression of p53 while decreasing the expression of c-Myc. Panel A: 1 hr stimulation of PC12 cells with 20 µM Fsk alone or in combination with a 1 hr pre-treatment with 100 µM EGTA and the effect on the expression of AnxA2, c-Myc and p53. The controls are indicated with black columns and are untreated cells with vehicle (DMSO) for Fsk treatment alone. For Fsk treatment with pre-incubation with EGTA, the control cells were treated with vehicle and EGTA. Panel B: Representative Western blots of the results shown in Panel A. GAPDH was used as a loading control. Panel C: The effect of 1 hr Fsk treatment on the phosphorylation of CREB and the expression of c-Fos. Untreated controls are shown as black columns and Fsk-treated cells with green columns. Representative Western blots are shown below the columns. Panel D: The effect of 1 and 2 hr Fsk treatment on the Ser25 phosphorylation of AnxA2. Proteins (12 µg) derived from total lysate from PC12 cells were subjected to 4–20% SDS-PAGE and western blot analysis. The results from three independent experiments (n = 3) are shown and were normalized to GAPDH. The standard deviations are also indicated. Statistical significance compared to control was determined by the two-tailed Student’s t-test (*p < 0.05)
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Frontiers in immunology
January 01 2022

Fig 2: C. difficile toxins increases the levels of phosphorylated CREB, which are involved in EGCs death induced by these toxins in vitro. (A) Representative Western Blot images showing phosphorylated CREB and tubulin (loading control) protein expression. (B) levels of pCREB (mean ± s.e.m.) measured by ELISA in EGCs challenged with TcdA and TcdB for 4h. (C) Cell death analyzed by RealTime-Glo annexin V apoptosis assay (mean ± s.e.m, n=6) in enteroglial cell (EGC/PK060399) challenged with TcdA and TcdB for 18 h in the presence and/or absence of 0.5 µM 666-15 (a CREB inhibitor) and 300 µM ATL313 (an A2a-selective adenosine receptor agonist), added one hour prior to C. difficile toxin challenge. (B, C) One-way ANOVA followed by Turkey test was used. These experiments are from two different replicates. ***p<0.0001, **p<0.001.
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Frontiers in immunology
January 01 2022

Fig 3: Regulation of IL-6 and S100B expression by adenosine receptors in EGCs challenged with C. difficile toxins. (A, B) Analysis of IL-6 gene expression (mean ± s.e.m) by qPCR in enteroglial cell (EGC/PK060399) challenged with TcdA and TcdB for 18h in the presence or absence of (A) 300 µM ATL313 (an A2a-selective adenosine receptor agonist), and (B) 100 µM ATL801 (an A2b-selective adenosine receptor antagonist). (C, D) Analysis of S100B gene expression (mean ± s.e.m) by qPCR in enteroglial cell (EGC/PK060399) challenged with TcdA and TcdB for 18h in the presence or absence of (C) 300 µM ATL313 (an A2a-selective adenosine receptor agonist), and (D) 100 µM ATL801 (an A2b-selective adenosine receptor antagonist).These experiments are from two different replicates. (A–D) One-way ANOVA followed by Turkey test was used. (E) Proposed model of the role of adenosine receptors on deleterious effects induced by C. difficile toxins on EGCs: C. difficile toxins (TcdA and TcdB) upregulate A2A and A3 and downregulate A2B receptors in EGCs. Activation of A2A and A3, as well as inhibition of A2B lead to inhibition of caspase 3/7 activation and consequently inhibits cell death induced by C. difficile toxins in a manner independently of PKA-CREB signaling. While A2A modulates S100B negatively, A2B promotes a positive effect in the expression of this mediator in EGCs challenge by C. difficile toxins. While the modulation of IL-6 expression by adenosine receptors in EGCs challenged by TcdA and TcdB seem to be complex, A2A decreasing its expression and A2B showing an opposite effect.