THE END?

Basically the following describes what I did over the summer. In addition to this summary, I learned how to do the entire Western Blot process and got introduced to chIP process. It was an unforgettable summer with so much skill learning. It’s unreal how much I benefited. I know I will carry these invaluable skills with me for the rest of my life especially as I look forward to conducting research in medicine and diseases. COULD NOT BE MORE GRATEFUL for what I have received… I will make it count. Thank you.

The aim of the project was to first optimize the immufluorescence (IF) protocol to image HeLa cells using varying dilution of dyes (Phalloidin, DAPI), dilution of primary antibodies (CHIP, EIF4a, VCP, Hsp90, p23), and Heat Shock (HS) duration (30 minutes, 90 minutes) in order to qualitatively determine the localization of several proteins specifically carboxy terminus of Hsc70 interacting protein (CHIP) and valosin-containing protein (VCP) with confocal microscopy. Using this visualization technique provided a lot of information since we were able to label proteins to observe their localization at different cellular environments (normal conditions, heat shock, and recovery). Previous investigations with HEK cells and Western Blot indicate that CHIP and VCP translocate to the nucleus upon HS. During this localization study (IF study) using HeLa cells it was found that there is a lot of CHIP and VCP present in the nucleus at the basal conditions (no HS). This could be due to CHIP translocation depending on cell cycle stage, some other stress on the cells, or just due to the different cell type (HeLa vs HEK) used in the experiments. Thus, a conclusion could not be reached qualitatively since insufficient data were gathered for VCP and since there were inconsistent results for CHIP. Although a conclusion cannot be attained, this study may still elucidate data about the localization of proteins in the nucleus if the images are further analysed quantitatively. Furthermore, two additional experiments were conducted to observe how the following conditions impacted localization of CHIP: (1) incubating HeLa cells with Hsp90 inhibitor and heat shocking cells for 30 minutes and (2) using media of previously heat shocked cells (90 minutes) to incubate cells in basal condition for 30 minutes. For both conditions an increase in amount of CHIP was observed in the nucleus consistent with heat shock conditions. The first result suggests that the CHIP translocation may be independent of Hsp90. The second result suggests that there could be possible cell signalling between HeLa cells (homogeneous cell culture) with some extracellular factor released during stress that may be detected by surrounding cells to induce the HSR.