Biological function of constitutively expressed sTNFR Fc The biological function of the secreted sTNFR Fc pro tein was evaluated and confirmed by three different selleckbio methods. First, a dot immunoblot assay was performed to determine whether the expressed sTNFR Fc was able to recognize TNF a. As shown in Figure 6, sTNFR Fc secreted from both HTB Inhibitors,Modulators,Libraries 11 and CHME 5 cells Inhibitors,Modulators,Libraries had the ability to bind to TNF a in vitro. Second, the ability of the sTNFR Fc to antagonize the toxic activity of TNF a was assessed by using TNF a sensi tive L929 indicator cells. In this case, an MTT assay was conducted to determine if the secreted sTNFR Fc protein was able to protect the test L929 cells from the cytotoxic impact of exogenous TNF a.
In this experi ment, L929 cells that were exposed to TNF a in Inhibitors,Modulators,Libraries the presence of the culture supernatant from non transduced control HTB and CHME 5 cells exhibited greatly reduced viability. In contrast, L929 cells that were exposed to TNF a in the presence of conditioned media from vector transduced HTB and CHME cells were protected from cell killing. Control studies confirmed that cells exposed to TNF a alone underwent high levels of cell death, while cells exposed to TNF a in the presence of 160 ng mL of commercially avail able, recombinant sTNFR Fc were strongly protected. These data indicate that the sTNFR Fc secreted from vector transduced cells mediated a significant cytoprotective effect, reflec tive of its ability to neutralize the biological activity of TNF a. The purified rTNFR mediated a slightly higher level of cytoprotection compared to 1,10 diluted conditioned medium from vector transduced HTB and CHME cells.
This reflects the higher concentration of purified Inhibitors,Modulators,Libraries rTNFR in this experiment, when compared to the level of sTNFR Fc present in 1,10 diluted cell culture supernatants from the vector transduced cells. As expected, conditioned medium from the control lentiviral Fc vector transduced cultures had no protec tive effect on the L929 cells Inhibitors,Modulators,Libraries exposed to TNF a. Similarly, sTNFR Fc expressed from the trans duced macrophages and neuronal cells was able to protect normal HTB 11 cells from TNF a mediated toxicity and transduced HTB 11 cells expressing sTNFR Fc were also protected from TNF a mediated toxicity.
To evaluate the ability of the secreted sTNFR Fc pro tein to block HIV 1 Tat mediated neurotoxicity, selleckchem Sorafenib primary rat neurons were treated with recombinant HIV 1 Tat protein in the presence or absence of conditioned medium from vector transduced cells, and cellular apop tosis was then measured 24 hours later by TUNEL assay. For this experiment, only culture supernatants from transduced HTB 11 cells were tested, since they contain roughly 5 fold higher levels of sTNFR Fc expression as compared to supernatants from trans duced CHME 5 cells.