Light the Night Walk and detecting EZH1 in patient cells

Hello again! My previous post discussed how I finally had a specific antibody to EZH1 in a cell line (HEK293). As a follow up, I wanted to determine if we could detect EZH1 expression in patient cells. I’m happy to report I was able to detect the EZH1 protein in patient cells (Figure 1). Experimental Read More …

Finally finding an antibody to detect EZH1 protein expression

Hello again! As a follow up to my previous post,I’ve continued the search for an antibody to detect EZH1. I’m happy to report that I finally found one. I was able to detect a band in HEK293 cells using the Cell Signaling Antibody. Using knockdowns targeting EZH1, I was able to determine that the band Read More …

Follow up to finding an antibody to detect EZH1 protein expression…the search continues

Following up on my previous post, I tested two antibodies I was planning on trying from Abcam and Novus that were used in previous publications. This time I tested directly in patient sample lysates and either saw no band, or many nonspecific bands (Figure 1). I have since reached out to the Orlando lab who Read More …

(Not quite) finding an antibody to detect EZH1 protein expression

Hello again! Here is my first attempt to find an EZH1 antibody. I have mainly focused on EZH2 in the PRC2 complex as the methyltransferase responsible for the H3K27me3 mark. However, EZH1 can functionally substitute for EZH2 in PRC2. We have a good antibody to detect EZH2 levels in patient AML samples. However, for some Read More …

Optimizing methodology for the detection of H3K27me3 levels using flow cytometry

Hello again! In previous posts I talked about how we were using a co-culture system to grow our leukemic cells. Although this is a great way to maintain cells, it can complicate some analyses where we cannot distinguish mouse (stroma) from human (AML) cells. We wanted to determine H3K27me3 levels (the mark that EZH2 is Read More …

Determining the plasticity of CD34 expression

Previously, we established that OCI-AML-20 requires stroma to maintain CD34 expression (https://zenodo.org/record/1187083#.Wr5-1C7wbIV). In this experiment, we wanted to see whether those cells grown in suspension are able to regain CD34 expression when cells are reintroduced to stroma. We find that OCI-AML-20 regains CD34 expression when put back on to stroma (either MS5 or OP9). Full Read More …

Determining CD34 expression in the presence and absence of stroma

We are currently working on a manuscript to characterize one of the cell lines that we established from growing patient samples. I previously discussed how we established OCI-AML-20 and I’m currently working on some experiments to complete the manuscript. One of those experiments is determining whether the CD34 expression seen in OCI-AML-20 relies on the Read More …

Western Blot of NSD3 in AML Cell Lines

In order to study NSD3 in the context of acute myeloid leukemia, I first need to determine the best reagents/tools. This includes characterizing model cell lines as well as antibodies that can be used for future experiments. Here I looked at the expression of NSD3 by western blotting in three AML cell lines using a Read More …