Katie Munechika / Dr. Ethel Cesarman - Week 7

 This week, I confirmed the IP results with western blots. For each IP, I took three samples for running in the western: input (proteins extracted from the cell lysate), unbound (proteins that did not bind to the antibody-coated beads after overnight incubation), and elution (proteins that bound to the beads and were eluted off of them after overnight incubation). On the western blot image, I was able to see elution bands for the NSD2 and STAT3 IP’s, however I did not see H1 bands in the elution lanes. This means that I was successful in pulling down (i.e. capturing on the beads) the NSD2 and STAT3 proteins, but was not able to capture the H1 isoforms in complex with these target proteins, even though we excepted them to be interactors. The IP’s for H1C and H1E did not work, as there were no elution bands. However, I know that these proteins were present in the samples because there were bands in the input and unbound lanes. Overall, these results imply that all proteins of interest were present in the cell lysate, but only NSD2 and STAT3 were able to be captured by the beads. Considering that the antibodies I used in the IP are the same as the ones that I used for the western, I believe the source of the problem is that the antibodies for H1C and H1E did not conjugate to the beads properly. Without the antibodies attached to the beads, the beads would not be able to capture the proteins, which would explain our results. Regarding why we do not observe H1 in the successful NSD2 elution, another issue that may have occurred is disruption of the protein interactions. I incorporated a sonication step into the cell lysis protocol because in my past experience, the mild detergent used in the lysis buffer is often not enough to break the nuclear membrane as well as the cell membrane. That being said, I have never worked with this particular lymphoma cell line before, so perhaps the sonication was too harsh and could have disrupted the interaction complexes.

I continued shadowing Dr. Morales this week at the Infectious Diseases clinic. One case that I found interesting was a returning patient that visited for a check-up after being treated for a surgical infection they received from a mastectomy procedure. At this point, they had finished the round of antibiotics that had previously been prescribed and had been off of antibiotics for about 3-4 weeks. When examining the site where the incision had been made during surgery, it seemed that the wound had closed properly but there was a lump nearby that could potentially be some fluid build-up. It was difficult for Dr. Morales to tell just from the physical exam if the infection was properly treated, so she recommended further imaging of the site. Additionally, she did not want to keep the patient on antibiotics for too long, so while they were in a stable condition, she also wanted to consult with the patient’s surgeon and oncologist. While observing these different cases, I have noticed how important imaging technology is in allowing for more precise and individualized treatment of each patient. I have also seen how crucial it is for clinicians in different fields to be able to communicate with each other and work together to determine the best treatment options for patients that have multiple simultaneous medical issues, which is extremely common for older patients especially.