I\'ve never had to interpret electrophoporation graphs and gels before and I was

Question

I've never had to interpret electrophoporation graphs and gels before and I was wondering if someone could help me understand the following results from a paper.

This is the actal papers link: https://academic.oup.com/hmg/article/23/1/69/722169

and these are the graphs I need to be able to explain:

The caption under it is:

Figure 1. Expression of recombinant API4 in TA muscle by electroporation. (A) Upper: 9-days after electroporation of API4 plasmid in WT and mSCS TA muscle there was strong expression of API4 protein in TA western blots. No API4 protein was detected in WT, mSCS and mSCS with electroporation of pcDNA3 as control (mSCS-pcDNA3). Lower: relative protein levels of API4 (# = normalized to GAPDH) in API4-electroporated WT (WT-API4) and mSCS (mSCS-API4) TA muscle (0.79 ± 0.03 and 0.74 ± 0.05, respectively) were significantly greater than the undetectable levels in untransfected muscle. n = 3; ***P < 0.001, Student's t-test. (B) Upper: API4, immunostained with antibody to API4 (green) was not co-localized with AChRs labeled with TxR-BT (red) but predominantly surrounded subsynaptic DAPI-labeled nuclei (blue) in API4-electroporated WT TA muscle (WT-API4). However, API4 labeling was much more prominent in subsynaptic nuclei in API4-transfected mSCS muscle (mSCS-API4) than WT muscle. Lower: 42.2 ± 4.9% of endplates labeled for API4 in mSCS-API4 mice, while compared with 6.3 ± 0.5% in WT-API4 mice. Scale bar = 15 µm; n = 7; ***P< 0.001, Student's t-test.

9-day after electroporation mSCS- WT mSCS API4 API4 WT mSCS PCDNA3 API4 GAPDH 0.5 WT mSCS mSCS T mSCS- pcDNA3 API4 API4 WT mSCS AChR/API4/DAPI 50 40 30 20 10

Explanation / Answer

This study is about various pathways which lead to neuromuscular junction (NMJ) degeneration in Slow-channel syndrome (SCS) and also how this kind of DNA damage and degeneration can be selectively blocked. To explore and observe the results, all the experiments have been carried out on mice models. Three groups of mice models were taken; the wild type (WT), the transgenic mSCS type and the mSCS-pcDNA3 as control. Transgenic mice model (mSCS) had been developed according to the experimental needs that exhibits all the clinical, electrophysiological and pathological features of the SCS disease.

Also, the study shows the effect of a protein, human apoptosis inhibitor 4 (API4) on TA muscle which has dimisnished subsynaptic DNA damage in mSCS to a certain extent.

Both the wild type and the transgenic mSCS type of mice were electroporated with plasmid vectors expressing recombinant API4 into the limb muscle. The mSCS-pcDNA3 mice were taken as control.

Results were observed after nine days of electroporation.

TA western blots were run to detect and analyse the protein expression in the muscle of both treated and untreated mice. By western blotting, the proteins were transfered from the gel to the membrane and then vizualised.

Diagram A:

First membrane marked API4:

After nine days of eletroporation, there has been strong expression of API4 protein in the TA western blots of both treated groups of mice i.e, WT-AIP4 and mSCS-AIP4 types.

But the untreated WT, mSCS and the control mSCS-pcDNA3 showed no API4 expression in the western blot.

Second membrane marked GAPDH:

The realtive expression of GADPH has been shown. Here all the treated and the untreated groups of mice showed almost similar GADPH expression which meant that API4 treatment had no such effect on the normal expression of other proteins.

The lower graph based on API4 expression level:

Relative protein levels of API4 in API4-electroporated WT (WT-API4) and mSCS (mSCS-API4) TA muscle were 0.79 ± 0.03 and 0.74 ± 0.05, respectively.

Here, ± 0.03 and ± 0.05 are respective error bars. These values are significantly greater than the undetectable levels in untransfected muscle. n = 3; ***P < 0.001, Student's t-test (This means that the result is based on the means of three same experiments and the p value of the t-test is <0.001 and error is very less)

Diagram B:

Upper images:

The first image is of API4 traeted wild type variety and the second image is of API4 treated mSCS type. API4 has been immunostained with an antibody to API4 (green). It was observed that the antibody did not co-localize with AChRs labeled with TxR-BT (red) but mostly surrounded subsynaptic DAPI-labeled nuclei (blue) in API4-electroporated WT TA muscle (WT-API4).

Also, API4 labeling was much more prominent in subsynaptic nuclei in API4-transfected mSCS muscle (mSCS-API4) than WT-API4 muscle.

This infered that there is extensive accumulation of recombinant API4 protein in the syubsynaptic nuclei region and thus the presence of greater amount of activated caspases, 3, 7 and 9 in the mSCS transgenic mice muscle than in the treated wild type.

Graph below:

This graph tells us about the % of API4-labelled endoplates in both the API-treated mice variety.  Percentage of API4-labelled endplates in mSCS-API4 mice was 42.2 ± 4.9%, while that in WT-API4 mice was 6.3 ± 0.5%.

Here, ± 4.9 and ± 0.5 are the respective error bars.

Scale bar = 15 µm; n = 7; ***P< 0.001, Student's t-test. (This means the test has been carried out seven times and the result values are based on the means of all the seven tests.)

This graph infered that the % of API4-labelled endplates is significantly greater (almost 7 folds) in treated mSCS mice compared to that of treated WT type.

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