staining, a thorough understanding of proper sterile techniques is needed. Sterility refers to cell culturing experiments without any contamination, therefore all of the experiment was performed under the hood. No discoloration signified no contamination. The entire experiment proved successful upon identifying the YFP dopamine followed by colocalization and concluding with a release of calcium in the cell. Introduction Structure and function of the dopamine transporter The dopamine active
Validating the candidate kinases phosphorylating at S13/S16 and S116 Kinase gain of function (GOF) analysis: The hit cDNAs of kinases will be co expressed along with cDNAs Htt exon 1, Htt 171 and Htt 586 containing 16Q-23Q in HEK 293 cells and the Phosho S13/s16 and S116 Htt will be examined using the appropriate antibody, Phos –tag SDS-PAGE method (Bustamante et al., 2015) also method of IP of Htt and mass spectrometry. Kinases will also be co expressed with appropriate S to A variants of the above
phosphorylation of the S869-V893 region of mGluR1a when expressed in HEK cells (Iacovelli 2003, Sallese 2000, Mundell 2003). These agonist-induced internalization processes are -arrestin 1/2 and dynamin-dependent (Mundell 2001, 2002, 2003). -arrestin 1 appears to be important in mGluR1 endocytosis; however, the agonist-stimulated internalization of mGluR1a is observed only when -arrestin 1 is co-expressed with either GRK2 or GRK5 in HEK cells. GRK2, GRK5, -arrestin 1, or -arrestin2 individually has no
Examine the Interplay between O-GlcNAcylation and phosphorylation on Sec24C/D in cell cycle progression and vesicle trafficking. Aim1: Determine how O-GlcNAcylation of Sec24C/D affects COPII vesicle secretion under normal and ER stress conditions. Research design: I previously identified nine O-GlcNAc sites on Sec24C. This was accomplished by expressing myc-6xHis tagged Sec24C in human embryonic kidney 293T cells treated with Thiamet-G, an OGA inhibitor, and glucosamine to increase O-GlcNAc occupancy
levels of specific trace metals have been suggested to contribute to the abnormal cellular phenotype, as well as the progressive cell degeneration associated with TRPML1 dysfunction4. The permeability of Zn2+ to TRPML1 suggests that this ion channel may play a role in Zn2+ regulation. A recent investigation of cytoplasmic Zn2+ accumulation in TRPML1 dysfunctional cells suggests the possibility that this protein may normally function to transfer Zn2+ from the lysosome to the cytoplasm5. Other studies
Rluc-LC3-assay can be used to estimate autophagic flux at a single time point by defining the luciferase activities in cell extracts. Moreover, the use of a live cell luciferase substrate makes it possible to monitor changes in autophagic activity in live cells in real time. This method has been successfully used to identify positive and negative regulators of autophagy from cells treated with microRNA, siRNA and small molecule libraries.242- Farkas T, Hoyer-Hansen M, Jaattela M. Identification of
Abstract: This lab was conducted to determine the efficiency of transfection in HEK293 cells using cell culture and lipofectamine by growing the HEK293 cells out in growth medium, and then passaging them into 24 well plate at 50% confluency. After the passaging of the cells, and aspirating the media after letting the cells grow in the wells for 24 hours, then a master-mix of lipofectamine and GFP plasmid DNA was added to 500 microliters of stock DMEM, and then adding 100 microliters of the master-mix
challenging due to the low-affinity, sub-stoichiometric and transient nature of the modification. Using a synthetic O-GlcNAc analog containing a diazirine photocrosslinking moiety (GlcNDAz), we are able to label native O-GlcNAcylated substrates in live cells and crosslink them to nearby glycosylation-mediated binding partners upon UV treatment 13. In experiments performed by Abhisheck Chhetri, Tim Smith and Thomas Meister, the Boyce lab showed that endogenous and recombinant Myc-6xHis-tagged Sec24C exhibit
and Student’s t-test was used for post-hoc analysis between vector and Dys-siRNA. (E) Quantification for YFP-Drp1 puncta on mitochondria; paired t-test was used to compare the same cells before and after stimulation. (F) Quantification for YFP-Drp1 puncta on mitochondria; paired t-test was used to compare the same cells in the vector and the Dys-siRNA group for before and
protein 1 (LMP1) is expressed in most EBV-associated cancers and it is well established that LMP1 is a major viral oncogene. Expression of LMP1 alone is sufficient to transform cells and recombinant EBV lacking LMP1 is incapable of immortalizing B-cells in vitro. Moreover, transgenic mice expressing LMP1 behind a B-cell specific promoter develop lymphomas. Exosomes are a population of small (40-150 nm) endocytically-derived extracellular vesicles produced from inward budding events on the limiting