SUMOylation Regulates BmNPV Replication by Moderating PKIP Intracellular Localization
Round 1
Reviewer 1 Report
This study analyzed the biological significance of SUMOylation in Bombyx mori nucleopolyhedrovirus (BmNPV) infection. SUMOylation of PKIP plays a key role in BmNPV replication. There are several potentially interesting observations in the manuscript by Xudong Tang and coworkers. The research is well planned and the text is logically described. After revision, this manuscript can be published in Processes.
1. There are NOT panel symbols (ex. A~D) in Figure 1 and Figure 6.These help to explain the figures.
2. There are NOT asterisks in Figure 2. Is this right? For instance, I thought there are significant differences between control and siRNA in panel (D). If this is right, it might need to show no significant difference (n.s) between control and siRNA.
3. The scale bar in Figure 3 is too small. You need to add a more visible scale bar.
4. In Figure 4, you showed SUMOlylation by using western blotting. SUMOlylation was significantly reduced by mutation of K70 but not k67 in PKIP. Were they performed in triplicate? Can you put the bar graph by quantification of western blot images?
5. There is NOT a scale bar in Figure 5. You need to add it.
Author Response
- There are NOT panel symbols (ex. A~D) in Figure 1 and Figure 6.These help to explain the figures.
Answer：Panel symbols were very helpful for the understanding of figures. Thank you for your kind comment. So, the panel symbols have been added to Figure1 and 6 in the revised version.
- There are NOT asterisks in Figure 2. Is this right? For instance, I thought there are significant differences between control and siRNA in panel (D). If this is right, it might need to show no significant difference (n.s) between control and siRNA.
Answer：We re-analyzed the original qRT-PCR data, and calculated the P value between control and siRNA group. And then asterisks were added on Figure 2.
- The scale bar in Figure 3 is too small. You need to add a more visible scale bar.
Answer: There are bars in the original Confocal pictures. While in the process of image processing, the bars become unclear because the graph becomes smaller and smaller. Clearer bars and description have been added to the revised Figure3.
- In Figure 4, you showed SUMOlylation by using western blotting. SUMOlylation was significantly reduced by mutation of K70 but not k67 in PKIP. Were they performed in triplicate? Can you put the bar graph by quantification of western blot images?
Answer: The IP and western blotting has been tried for several times. And the results were consistent. The bar graph by quantification of western blot images will help to present the results in better way. An internal reference may need to normalize the quantification data. Since our proteins sample were obtained from IP, we are not sure about the reference protein. Sorry for that.
- There is NOT a scale bar in Figure 5. You need to add it.
Answer：Sorry, the unclear pictures embedded in the paper have caused inconvenience to you. In the original pictures we submitted, Figure 5 contains clear bars.
Reviewer 2 Report
The manuscript by Shen et al. describes an interesting discovery about the SUMOylation of PKIP in BmNPV. The overall draft is well written. It is very easy to follow and was a pleasant read. All the experimental designs are solid. Conclusions are solidly supported by obtained results. This reviewer supports its publication after a single concern is addressed.
1. The mutagenesis analysis is a relatively indirect way to confirm K70 is the SUMOylation site. In order to definitely confirm the SUMOylation at K70, it is much better to use the mass spectrometry-based fingerprinting to definitively assign it. So an MS-MS analysis of SUMOylated-PKIP is recommended.
Author Response
- The mutagenesis analysis is a relatively indirect way to confirm K70 is the SUMOylation site. In order to definitely confirm the SUMOylation at K70, it is much better to use the mass spectrometry-based fingerprinting to definitively assign it. So an MS-MS analysis of SUMOylated-PKIP is recommended.
Answer: That is true that the mass spectrometry-based fingerprinting is the direct way to tell if a protein was modified by SUMO or other factors. Our data proved the PKIP was modified by SUMO in an indirect way. Due to the limitation of our experimental platform and the time left for us, and with the coming of the Spring Holiday (the students also will leave school for a long time. Kind of short of manpower), we are sorry that we can't further improve our results with the method you kindly suggested at present.
