EEF1AKMT4 trimethylates eEF1A2 at K36 site in GBC. (IMAGE)
Caption
(A) Spatial distribution of the five main methylation sites in the protein structure of eEF1A2 combined with GDP (PDB: 6ra9). Most of the methylation sites were located in the nucleotide-binding domain of eEF1A2. (B) Relative mRNA expression of five methylases, EEF1AKMT4, METTL13, N6AMT2, METTL21B, and METTL10, which are responsible for methylation of K36, K55, K79, K165, and K318 sites, respectively, was quantified by qRT-PCR in 10 cases of gallbladder cancer tissues and their patient-paired normal tissues. (C) Coomassie blue staining of anti-eEF1A2 co-immunoprecipitation in GBCSD, SGC996, and HEK293T cell lines. Target bands are indicated by a black box and were cut and subjected to LC-MS/MS to analyze the methylation status. (D) Methylation status at the K36, K55, K79, K165, and K318 sites of eEF1A2 in GBCSD, SGC996, and HEK293T cell lines were analyzed. (E) Representative tandem mass spectra identifying in vitro tri-methylated(upper) and non-methylated (lower) eEF1A2K36. m/z for b and y ions observed in the spectra are indicated in red and blue, respectively. (F) Histogram showing the methylation changes in K36, K55, K79, K165, and K318 before(upper) and after(lower) knockdown of EEF1AKMT4 in GBCSD cells. (G) Selected ion chromatograms for non-, mono-, di-, and trimethyl eEF1AK36 peptides from GluC digestion of endogenous eEF1A2 immunoprecipitated from whole-cell lysates of GBCSD, indicating the methylation status shift after EEF1AKMT4 knockdown. (H) Western blot analysis of the knockdown efficiency of EEF1AKMT4 in GBCSD and the status of eEF1A2 and eEF1A2K36me3. (I) Expression of eEF1AKMT4 and eEF1A2 K36me3 levels in GBC tumor tissues and paired normal tissues were examined by western blotting and analyzed using ImageJ. Statistical significance between groups was assessed using Student’s t-test. ns, not significant; *, p < 0.05; **, p < 0.01.
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Genes & Diseases
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