MS/MS analysis identifies S861 as a low level phosphorylation site.

<p>A) Venn diagram of phosphorylation sites identified in 3 replicates of MS/MS analysis <i>of in situ</i> auto-phosphorylated HAE. T851 is marked by a red box to indicate it was mutated in the protein analyzed in the 2nd and 3rd replicate, precluding its identification as a phosphorylation site. S861 is marked in blue to indicate the 2nd and 3rd replicates were conducted in order to identify this site. B) <i>In situ</i> auto-phosphorylation levels of activation segment mutants. N = 4 biological replicates. Error bars represent standard deviation. Statistical tests between the single mutants were carried out by a 2 sample t-test. Statistical tests to compare the 4 activation segment double mutants was performed by ANOVA with Tukey-Kramer correction for multiple comparisons. C) Representative stains for phosphorylation and total protein by Pro-Q Diamond and coomassie blue, respectively.