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. Author manuscript; available in PMC: 2023 Jan 19.
Published in final edited form as: Kidney Cancer J. 2022 Nov 18;6(3):179–193. doi: 10.3233/kca-220009

Fig 2. Loss of SETD2 expression results in loss of eEF1A1 methylation.

Fig 2.

In order to validate MaxQuant peptide quantification, further analyses were conducted. The extracted ion chromatograms (EIC) were manually analyzed. The ratio of the a-b) eEF1A1_K318 peptide intensity was calculated in the WT and SETD2-KO lysates (AUC of the grey zones compared; vertical gold line corresponds to retention time at which the peptide was originally identified). c-d) EICs were also analyzed for the peptide eEF1A1_K165 WT/KO ratio. e) The ratio of eEF1A1_K165 was compared in SILAC-labelled lysate again (replicates “C” and “D”). However, instead of enrichment via immunoprecipitation with anti-Kme(1–3) antibody, the eEF1A1 protein was cut from a gel (i.e., enrichment based on protein size). f) The six above replicates were included for a pooled analysis, which demonstrates an increase in the WT/KO ratio for the K165me3 peptide relative to the K165me2 ratio. g) Synthetic peptide labeled with heavy arginine (13C, 15N) was formulated for eEF1A1_K165(me3). Synthetic peptides were spiked into WT and SETD2-KO lysates for HKC and 786-O, allowing for relative quantification of peptide in non-SILAC-labeled systems.