exam 1 Hayashi article Flashcards
What did previous studies determine about phosphorylation of GluA1 and what was the purpose of Hayashi group’s research?
It was assumed that phosphorylation of GluA1 receptors is implicated in LTP. Previous studies used antibodies against phosphoisoforms of gluA1 receptors that only showed the presence of phosphorylation, and not how much is phosphorylated. Hayashi group wanted to quantify the amount of phosphorylated receptors to unphosphorylated receptors.
What is, and how do you do transfection, SDS PAGE and western blot?
Check notes.
What happened when the researchers performed a western blot with Okadaic acid?
Phosphorylation increased in the presence of Okadaic acid. This shows that there is phosphorylation happening at the GluA1 site.
What is the limitation of their western blot and SDS PAGE they performed with Okadaic Acid?
It only shows the presence of phosphorylation and not the proportion of phosphorylated GluA1 receptors relative to the unphosphorylated GluA1 receptors.
What method did they use to detect phosphoisoforms and why did they need to do this?
They used Phos Tag SDS Page to detect phosphoisoforms. They had to use it because SDS Page does not show the individually phosphorylated sites, as phosphate is too small to be detected by the SDS Page.
How does phos-tag SDS PAGE separate proteins?
It separates them based on the amount of phosphorylation and the location of phosphorylation on the glua1.
What results did the phos-tag SDS PAGE show in the presence and absence of Okadaic Acid? How did the results change when they added lamdaphosphatase and why is this phosphatase necessary?
In the absence of OA, the band looked like the band in regular SDS PAGE, which is expected. Adding OA showed multiple bands. This means that there are specific phosphorylated sites on the GluA1 receptors. They added lamdaphosphatase, which overpowers the OA. Adding lamdaphosphatase with OA shows only one band, which means that the bands on phos-tag SDS PAGE are only due to phosphorylation.
What does SDS PAGE show with and without OA?
There is only one band that travels about the same distance in the presence and absence of OA.
How did the researchers determine which bands in phos tag SDS PAGE correspond to which phosphorylated serines? What did the researchers conclude overall?
They generated 4A mutated receptors under maximally phosphorylated conditions by adding forskalin and forbol ester (mechanisms in notes). Then added antibodies specific to each phosphoisoform. Slide 13. They concluded that phosphorylation is due to 3 sites on the GluA1 receptors: S831, S845, T840.
What did the researchers do to determine double phosphorylation and what did they conclude?
They used 3A mutants to run their phos-tag SDS PAGE on. They used 4A mutants as method of comparison. In two combinations- S831/S845 and S831/T840, they saw a band that migrated slower than their individual phos-tag SDS PAGE results. This was not present in S845/T840 phos-tag SDS PAGE. The higher band corresponded to double phosphorylation. Therefore, they used phosphospecific antibodies to conclude that these two sides are doubly phosphorylated.
Walk through figure on slide 16.
Why is it important that the C terminus Ab binds to all phosphoisoforms equally, how did the researchers confirm equal affinity of C terminus Ab, and what did they ultimately conclude?
It is important that the antibody against the C terminus binds to all phosphoisoforms equally because you want to use the western blot to determine the proportion of phosphorylated GluA1. They used a negative control, and a positive control with a hyperphosphorylating protocol where all GluA1 subunits were 100% phosphorylated. They concluded that the C terminus antibody binds to all phosphoisoforms with equal affinity.
Did the researchers conclude that the western blot labeling intensity is proportional to the amount of phosphorylated GluA1? What did they do to confirm?
Yes. They used ratios of control + OA to get proportions in the phos-tag SDS PAGE. So they had percents of phosphorylation. This was the densitometry. They confirmed densitometry results by using AQUA, which showed the percent of phosphorylated to unphosphorylated GluA1.
What did the researchers do to detect phosphoisoforms in rat hippocampi tissue? What did they conclude?
They used hippocampus, and immuniprecipitated the glua1 antibody with and without the lamdaphosphatase treatment. They ran the sample under SDS PAGE and phos-tag SDS PAGE. Phos-tag shows phosphorylation because the band goes away in the presense of the lamdaphosphatase. However, when they analyzed the density, they found very little proportion of the GluA1 phosphorylated.
Why did the researchers use a standard curvet to assess proportion of phosphorylation? How did the standard curve work? What did they conclude from the standard curve?
They got low percent of phosphorylation so they wanted to make sure that their densitometry was working accurately. They phosphorylated the sites to 100%, and used proportions to dilute them. Their line graph showed the percent phosphorylation and densitometry of GluA1. The standard curve gave the same percents as the densitometry, which showed 1. that the densitometry method worked and 2. the proportions are actually that low.
Why isn’t using the whole hippocampus useful and what did they do instead? (what they did + method) What were the results when they did the phos-tag SDS PAGE?
Whole hippocampus is not helpful because it is only a fraction of the post synaptic density. They used a PSD-enriched hippocampus. PSD is more useful because GluA1 is present in the PSD.
Method: they used a centrifuge to separated the microsomes (pre-synaptic specialization), soluble fraction (everything else), and the PSD (post synaptic density). All of the bands were fast moving, which supported their previous conclusions that GluA1 receptors are not phosphorylated.
