On 2013 Jul 17, Toby Gibson commented:

Chk1 is an important DNA damage checkpoint protein kinase. Biochemical evidence suggests that the sites most vigorously phosphorylated by the Chk1/2 kinases match or are close to the pattern (LF)xRxx(pST)(LF). See, for example, these articles (PMID: 10648819; 10761933; 11821419; 12711320). If only one of the three specificity determining positions are lost, the kinases may still phosphorylate, but at reduced activity. This gradation in activity might have biological significance and also means that it is difficult to define motif patterns for Chk kinase substrate sites. Currently we are trying to do this for the ELM protein motif resource (http://elm.eu.org/).

In the paper that I would like to comment on here, the reported phosphorylation site at Ser47 of p73 - VgGtd(pS)S - lacks all of the strong specificity determinants (capitalised positions). It has been confounding our attempt to define a Chk phosphorylation site pattern for ELM. However, based on a close examination of the figures in the paper, we have now decided to exclude this p73 site from further consideration.

The MCB paper is open access and high resolution figures are available at both the MCB site and through PubMed.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC262369/figure/f2/

Fig. 2A lower left gel. Lanes 3-4 are the same as lanes 5-6. Note also the inconsistent gap length between band pairs. This gel appears to be a bounded grey box into which band pairs have been pasted.

Fig. 2B upper left gel. This appears to be a bounded grey box in which the + lanes have been pasted in. These have vertical edges that do not correspond to the edges of the bands. The - lanes are just smooth, grey blanks.

Fig. 2B lower left gel. The band in lane 4 is the same as in lane 5. Associated dots and shadows are diagnostic despite a slight size difference.

Fig. 2B lower right gel. This is a bounded grey box into which three bands have been pasted. The band in lane 1 has been pasted over the edge line! The band in lane 3 has also been pasted over the edge line!

Fig. 2C Lower left gel. The band in lane 2 has been pasted over the edge line! This is another grey box into which bands have been pasted.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC262369/figure/f3/

Fig. 3A upper gel. Bands in lanes 1-4, 6-7, 10 are identical. Maximum resolution is required to see this unambiguously.

Fig. 3B upper gel. Bands in lanes 4, 6, 9-10 are identical. Maximum resolution is required to see this.

Fig. 3B middle gel. Bands in lanes 2,4 and probably 3 are the same. Bands in lanes 6,7,9 are the same.

Fig. 3D upper right gel slice. All eight bands are identical. Maximum resolution is required to see this (it is not clear from the pdf download of the whole paper). Also these bands might be elongated versions of the reused bands listed for Fig. 3A.

Fig. 3 D lower left gel slice. Lanes 2-7 are identical to lanes labelled 8-13 of the lower right gel slice. According to the labelling above each lane, these are certainly to be understood as different experiments.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC262369/figure/f4/

Fig. 4A Upper right slice. Band pair in lanes 1-2 are the same as 7-8. Band in lane 4 is the same as band in lane 6.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC262369/figure/f5/

Fig. 5D lower panel. Bands 1-3 are the same as bands 6-8, except that they are the mirror image.

The paper is ten years old. Therefore it is unlikely that the raw data used to assemble these figures still exists, though it would be fascinating to make comparisons if any original data can be found.

On 2013 Jul 17, Toby Gibson commented:

Chk1 is an important DNA damage checkpoint protein kinase. Biochemical evidence suggests that the sites most vigorously phosphorylated by the Chk1/2 kinases match or are close to the pattern (LF)xRxx(pST)(LF). See, for example, these articles (PMID: 10648819; 10761933; 11821419; 12711320). If only one of the three specificity determining positions are lost, the kinases may still phosphorylate, but at reduced activity. This gradation in activity might have biological significance and also means that it is difficult to define motif patterns for Chk kinase substrate sites. Currently we are trying to do this for the ELM protein motif resource (http://elm.eu.org/).

In the paper that I would like to comment on here, the reported phosphorylation site at Ser47 of p73 - VgGtd(pS)S - lacks all of the strong specificity determinants (capitalised positions). It has been confounding our attempt to define a Chk phosphorylation site pattern for ELM. However, based on a close examination of the figures in the paper, we have now decided to exclude this p73 site from further consideration.

The MCB paper is open access and high resolution figures are available at both the MCB site and through PubMed.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC262369/figure/f2/

Fig. 2A lower left gel. Lanes 3-4 are the same as lanes 5-6. Note also the inconsistent gap length between band pairs. This gel appears to be a bounded grey box into which band pairs have been pasted.

Fig. 2B upper left gel. This appears to be a bounded grey box in which the + lanes have been pasted in. These have vertical edges that do not correspond to the edges of the bands. The - lanes are just smooth, grey blanks.

Fig. 2B lower left gel. The band in lane 4 is the same as in lane 5. Associated dots and shadows are diagnostic despite a slight size difference.

Fig. 2B lower right gel. This is a bounded grey box into which three bands have been pasted. The band in lane 1 has been pasted over the edge line! The band in lane 3 has also been pasted over the edge line!

Fig. 2C Lower left gel. The band in lane 2 has been pasted over the edge line! This is another grey box into which bands have been pasted.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC262369/figure/f3/

Fig. 3A upper gel. Bands in lanes 1-4, 6-7, 10 are identical. Maximum resolution is required to see this unambiguously.

Fig. 3B upper gel. Bands in lanes 4, 6, 9-10 are identical. Maximum resolution is required to see this.

Fig. 3B middle gel. Bands in lanes 2,4 and probably 3 are the same. Bands in lanes 6,7,9 are the same.

Fig. 3D upper right gel slice. All eight bands are identical. Maximum resolution is required to see this (it is not clear from the pdf download of the whole paper). Also these bands might be elongated versions of the reused bands listed for Fig. 3A.

Fig. 3 D lower left gel slice. Lanes 2-7 are identical to lanes labelled 8-13 of the lower right gel slice. According to the labelling above each lane, these are certainly to be understood as different experiments.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC262369/figure/f4/

Fig. 4A Upper right slice. Band pair in lanes 1-2 are the same as 7-8. Band in lane 4 is the same as band in lane 6.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC262369/figure/f5/

Fig. 5D lower panel. Bands 1-3 are the same as bands 6-8, except that they are the mirror image.

The paper is ten years old. Therefore it is unlikely that the raw data used to assemble these figures still exists, though it would be fascinating to make comparisons if any original data can be found.