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. 2014 Aug 15;127(16):3505–3520. doi: 10.1242/jcs.149112

Fig. 1.

Fig. 1.

Nucleoporin Tpr is phosphorylated in vivo at residues S2059 and S2094. (A) Schematic representation of the TprC and TprC-M4 constructs. (B) COS-1 cells transfected with constructs encoding FLAG–TprC or FLAG–TprC-M4 were metabolically labeled, and FLAG-tagged proteins were immunoprecipitated, resolved and autoradiographed. (C) In-vivo-labeled TprC and TprC-M4 were digested with trypsin, and the resulting phosphopeptides were mapped by 2D thin-layer chromatography (TLC). Arrows indicate the labeled phosphopeptides that were persistent in the in vivo map of TprC-M4. Dotted circles indicate ERK2-mediated phosphorylation, which is lost from TprC-M4. (D) Phospho-amino-acid analysis of the labeled TprC-M4 protein. The dotted circles show the migration of phosphorylated threonine (pThr) and phosphorylated tyrosine (pTyr) amino acid standards detected by ninhydrin staining. (E) Metabolically labeled TprC-M4-(S2046,2047,2049A), TprC-M4-(S2059A) and TprC-M4-(S2094A) proteins were digested with trypsin and were mapped by 2D-TLC. White arrows and dotted circles indicate the disappearance of labeled phosphopeptide spots that are indicated with black arrows for TrpC-M4.