OBJECTIVE: Logical function of Lipopolysaccharide (LPS)-Induced Factor (LIF), and to identify the factors involved in the LIF signaling pathway in response to LPS, using a new method (Hybrid) for protein detection. The hybrid method combines the main features of the ELISA and Western blot assay and provides the advantage of measuring multiple proteins simultaneously.
MATERIALS & METHODS: A culture of mouse RAW c cells in RPMI media supplemented with 10% FBS at 37°C in 5% CO2 was established. Polymerase Chain reaction (PCR) used specifically designed primers and DNA cloning of LIF. Cloned LIF cDNA was transfected into macrophages. Western blot, ELISA, and hybrid method analyses were used to determine production of proinflammatory cytokines.
RESULTS: LIF induced TNF-ɑ production during the initial time interval (1-12 hrs) and reduced TNF- ɑ production during the later time interval (10-24 hrs). LIF induces apoptotic proteins such as Caspase 1/3 at that time. The CASPASE 1/3 gene expression steadily increased.
The 7% Formaldehyde solution was suitable for concentration for cell lysate fixation, and was used in a 96-well microplate with more stringent washing conditions (shaking at 100-150
rpm), which can specifically detect an antibody without a false positive. A cell lysate concentration of 2 μg assured protein detection by the hybrid method. At least 20 minutes were needed for protein detection but 40 minutes was the optimal time.
CONCLUSION: There was a two-sided association between LIF and TNF-ɑ: LIF activates TNF-ɑ gene expression in early stages and suppresses its production in later stages by inducing the apoptotic protein, CASPASE 1/3. The hybrid method is suitable for protein detection in various types of cells. Usage of the hybrid method allows detection and normaliz intracellular and extracellular proteins. Factors such as P38, ERK1, and MCP-1 are involved in the LIF-dependent signaling pathway.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/49152 |
| Date | 14 August 2024 |
| Creators | Elghadafi, Radwa Taher |
| Contributors | Tang, Xiaoren |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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