Sordaria macrospora is a filamentous fungus that serves as very useful system for scientific research due to a short life cycle and easy manipulation. Just like any other model organism, it is important to have an effective loading control to validate experiments in the Sordaria macrospora. In addition, the growth and morphogenesis of filamentous fungi is dependent on actin organization. Actin is a very abundant protein across biological species and can transition between monomeric and filamentous states. Actin has an alpha and beta isoform, both of which share nearly 98% percent sequence homology with total actin. Actin recruits a variety of actin regulators in order to carry out many cellular functions, including cell division, cell motility, cell growth and differentiation, muscle contraction and more. The following articles use an actin antibody as a loading control in the study of Sordaria macrospora and its ability to survive in ambient air without carbonic anhydrases, as well as the role of autophagy in fruiting body development.
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Actin Antibody (mAbGEa) [NB100-74340] - Western blot analysis of Actin expression in 2) HeLa, 3) NTERA-2, 4) A431, 5) HepG2, 6) MCF7, 7) NIH-3T3, 8) PC-12 and 9) COS-7 whole cell lysates.
First, Lehneck et al used the actin antibody as a loading control in their research to further investigate how Sordaria macrospora have the ability to survive without carbonic anhydrases. To begin, it was established that Sordaria macrospora coded for a CAS4 carbonic anhydrase, which is expressed during early sexual development and regulated by CO2. Next, in order to confirm this expression, a CAS4-EGFP construct was created and measured in western blot. The western blot analysis showed that the signal from CAS4 activated EGFP secretion. These results were confirmed by the use of an actin protein against an actin antibody as the loading control. It was also found that deleted of CAS4 prevented normal fruiting body development. In the end, it was clear from their results that CAS4 is post transnationally glycosylated and secreted for a role in vegetative growth and germination.
In addition, Voigt et al used an actin antibody as a loading control in their research of whether the autophagy genes SmATG8 and SmATG4 were required for fruiting body development and vegetative growth. Since autophagy is under-studied in multicellular filamentous organisms, this group analyzed the impact of two key autophagic players in fungi development. Through the generation of homokaryotic deletion mutants it was discovered that autophagy does in fact play a role in these processes. Specifically, it was found that SmATG8 is terminally processed by smATG4, which was confirmed in western blot using an anti-EGFP antibody. The crude wild-type protein extracts alongside the actin antibody was used as a control. They also went on to find that EGFP-SmATG8 is localized to the autophagosome, whereas EGFP-smATG4 is located in the cytoplasm. This is important because smATG4 is necessary for proper autophagosome formation, so a recruitment process may take place.
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