Sds page vs western blot series#

Having established that JT11 selectively activates CB2R, we focused on its immunomodulatory potential. JT11 Induces ERK1/2 Phosphorylation through a CB2R-Dependent Mechanism in Jurkat Cells As a control, no significant increase in cleaved PARP levels was evident when the cells were treated with SR144528 alone ( Figure 3B). This data further supports the hypothesis of CB2R involvement in the activity of this novel compound ( Figure 3B). Furthermore, it is noteworthy that the pretreatment with CB2R antagonist SR144528 abolished the effect of JT11 on PARP, in fact the differences in the levels of cleaved PARP in the samples pretreated with SR144528 were comparable to the sample incubated with the vehicle alone. The results displayed a significant increase in cleaved PARP levels after treatment with 2 μM JT11 for 24 or 48 h, in line with the flow cytometric data of PI staining. To verify whether these results could be ascribed to apoptotic cell death, we run a Western blot analysis of the nuclear enzyme poly (ADP-ribose) polymerase (PARP), which serves as a marker of apoptosis, since it is cleaved by caspases during programmed cell death process. JT11 Effect on Cell Viability and Proliferation through a CB2R-Dependent Mechanism in Jurkat Cells Then, we tested the anti-inflammatory activity of this novel compound, evaluating its modulatory effect on both intracellular signaling cascade and the release of pro-inflammatory cytokines triggered by bacterial lipopolysaccharide (LPS) in peripheral blood mononuclear cells (PBMCs) from healthy donors. Importantly, we have also confirmed its ability to selectively activate CB2R. Of course, we performed preliminary experiments on Jurkat cell line to evaluate the impact of JT11 on cell viability and verify its potential cytotoxicity. Here, we evaluated in vitro the biological activity of this new synthetic, selective and high-affinity CB2R agonist with a focus on its potential CB2R-mediated anti-inflammatory role. One of these compounds, the N-(4-methylcyclohexyl)-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide, also called JT11 ( Figure 1), has been considered in the present work. More recently, additional derivatives have been developed with the aim of further increasing the affinity and selectivity for the CB2R.

Sds page vs western blot series#

described the design, synthesis and pharmacological properties of a series of derivatives of 1,8-naphthyridine-2(1H)-one-3-carboxamide, which have been shown to act as potent selective CB2R agonists. JT11 as CBR2 ligands could enhance ECS immunoregulatory activity and our results support the view that therapeutic strategies targeting CBR2 signaling could be promising for the treatment of chronic inflammatory diseases. Finally, it reduced LPS-induced ERK1/2 and NF-kB-p65 phosphorylation and pro-inflammatory cytokines release in human PBMCs, proving to possess in vitro anti-inflammatory properties. JT11 regulated cell viability and proliferation through a CBR2-dependent mechanism in Jurkat cells, exhibiting a mild pro-apoptotic activity. We confirmed selective CBR2 activation by JT11. Selective CB2R antagonist SR144528 and CBR2 knockdown were used to further verify the selectivity of JT11. Finally, we evaluated its impact on LPS-induced ERK1/2 and NF-kB-p65 activation, TNF-α, IL-1β, IL-6 and IL-8 release in peripheral blood mononuclear cells (PBMCs) from healthy donors. Its pro-apoptotic activity was investigated analyzing both cell cycle and poly PARP cleavage. Trypan Blue and MTT assays were used to test the cytotoxic and anti-proliferative effect of JT11 in Jurkat cells. Herein, we evaluated in vitro the biological activity of a novel, selective and high-affinity CBR2 agonist, called JT11, studying its potential CBR2-mediated anti-inflammatory effect. Targeted CBR2 activation could limit inflammation, avoiding CBR1-related psychoactive effects. The endocannabinoid system (ECS) exerts immunosuppressive effects, which are mostly mediated by cannabinoid receptor 2 (CBR2), whose expression on leukocytes is higher than CBR1, mainly localized in the brain.