## 3 Results
### Post-Translational Regulation of Transgenic GPD2 Protein  
To explore the possible involvement of other protein kinases in regulating glycerol accumulation under salinity stress, we also examined cells treated with the protein kinase inhibitor staurosporine. Due to its strong affinity for the ATP-binding site of kinases, staurosporine is a prototypical ATP competitor, but has very little selectivity [63]. To ensure that staurosporine was taken up by our _C. reinhardtii_ strains, we first tested that it did inhibit, as previously reported [64], the transcriptional induction of _HEAT SHOCK FACTOR 1 (HSF1)_ after a heat shock treatment (Figure 5A). Of particular relevance to this study, the incubation of cells with staurosporine also completely abolished the glycerol accumulation triggered by salt stress in both wild type and OX-12 strains (Figure 5B). However, the abundance of transgenic GPD2 protein was still reduced in OX-12 cells incubated with staurosporine to an analogous extent as in untreated cells (Figure 5C, HS+NaCl). Similar results were observed when cells were treated with both staurosporine and the translation inhibitor cycloheximide (Figure 5D), indicating that staurosporine is blocking the NaCl-triggered activation (or de-repression) of pre-existent transgenic GPD2 protein for glycerol synthesis, but not its change in stability. Figure 5: Effect of the non-specific protein kinase inhibitor staurosporine on glycerol accumulation by the wild type and OX-12 strains. (**A**) Semi-quantitative RT-PCR analysis of _HEAT SHOCK FACTOR 1 (HSF1)_ transcript abundance after exposing wild type and OX-12 cells to heat stress (40 \({}^{\circ}\)C for 20 min) in the absence or presence of staurosporine (STS). Amplification of the mRNA corresponding to _ACT1_ was used as a control for equal amounts of input RNA and for the efficiency of the RT-PCRs. The panels show representative reverse images of agarose resolved PCR products stained with ethidiumbromide. (**B**) Glycerol content in the wild type and OX-12 strains cultured in HS medium or HS medium containing NaCl, in the absence (Ctrl) or presence of staurosporine (STS). Values indicate the mean \(\pm\) SD of three independent experiments (n = 3). Different lowercase letters indicate a significant difference among means (one way ANOVA with post hoc Tukey HSD test, \(p\) < 0.05). (**C**) Immunoblot analysis of transgenic GPD2 protein levels detected with an anti-AcV5 antibody. OX-12 cells were cultured, for the indicated times, in minimal HS medium or HS medium containing 100 mM NaCl, either in media alone or in the presence of cycloheximide (+Chx), staurosporine (+STS) or both cycloheximide and staurosporine (+Chx+STS). Immunodetection of histone H3 was used as a control for equivalent loading of the lanes. (**D**) Glycerol content in the wild type and OX-12 strains cultured in HS medium or HS medium containing NaCl, in the absence (Ctrl) or presence of both staurosporine and cycloheximide (STS+Chx). These results suggested that a protein kinase(s) may play a role in the proposed post-translational regulation of transgenic GPD2 under salinity stress. However, it was unclear whether GPD2 itself and/or a protein(s) affecting GPD2 function might be the target of the putative kinase(s). We took advantage of the AcV5 epitope tag to partly enrich, by affinity purification, transgenic GPD2 protein from OX-12 cells cultured in HS medium or HS medium containing NaCl. These samples were examined by LC-MS/MS, as previously described [40], to identify differentially phosphorylated peptides. Unfortunately, GPD2 protein abundance was substantially reduced under salinity stress (as discussed above) making it difficult to purify enough protein to identify modified peptides with high confidence. In two independent experiments, a single phosphorylated peptide was detected exclusively in the NaCl-treated samples, albeit at only low medium confidence (Figure S3A,B).