MCD BIO 165A Lecture Notes - Lecture 3: Confocal Microscopy, Ctgf, Small Interfering Rna
11 Feb 2018
School
Course
Professor
Document Summary
Low mechanical stress --> yap/taz in the cytoplasm. Mechanical stress influences actin stress fibers which regulate yap/taz activity. Hippo signaling also regulates yap/taz activity but independently of mechanical stress. Uses confocal microscopy with immunofluorescence: measures yap/taz localization. Cell cell contact in this experiment --> contact inhibition. Negative regulators --> depolymerize f-actin --> decrease yap/taz activity. Exact pathway through which f-actin influences yap/taz activity is not clear *** Want to see if knockdown of negative regulators will increase yap/taz activity. Need to use an environment in which yap/taz are usually not activated --> soft matrix or small area. Fig 3: sirna screen for genes requires to prevent yap/taz activation on soft matrices. Ctgf and ankrd1 are transcriptional targets of yap/taz mrna expression levels measured by rt-pcr. S3a: used to make sure that the specific sirnas are targeting the correct proteins sico is a control to make sure sirna molecule isn"t toxic to cell and there are no off target effects.
