They asked how they could confirm the PDGF mediated anti senescence effects as well as how FGF 2 is involved in PDGF BB anti senescence activity. For this, they decided to further analyze the proteins within the mESC-CM through a western blot. They did this to further visualize the DNA samples.

p21 and p53, both of which play a role in senescence within the cell, were seen to have lower expression levels (Fig. 2H and 2G). p21 is not as clear or prevalent as the other samples within this photo. This can be seen under the CM2 column.

Western Blot; The Procedure

1. Western Blot Analysis is a technique widely used in molecular biology. The procedure occurs through protein denaturation (which was already done within real-time PCR), followed by gel electrophoresis.

2. Gel electrophoresis occurs through a medium, typically within an agarose gel, in which an electric current is ran, negative to positive. The idea is that since DNA is negatively charged, the current is ran to cause the DNA samples to traverse to the positive side.

3. A comb is utilized to create wells for each one of the samples.

4. The samples are then each pipetted into their respective wells, usually after being stained, to help with ultra violet visualization after the procedure.

Fig 2H. Expression levels of pRb, p53, and p21 through Western Blot Analysis.