Fig 5. Hit proteins from the in-cell ITDRCETSA experiments with thymidine.
(A) In-cell MS-ITDRCETSA curves of enzymes involved in the thymidine metabolism cascade. (B) Lysate-western blot (WB)-ITDRCETSA validation with dTXP. Lysates were treated with 10, 2.5, 0.6, 0.1 and 0 (control) mM of dT, TMP, TDP or TTP and probed for RRM1 and ABCF1 with actin as loading control. (C) In-cell MS-ITDRCETSA curves of ABCF1 and ABCF2. Data is presented as two individual technical replicates for each condition from 2 biological replicates (biological replicate 1 (dT_b1, blue) and biological replicate 2 (dT_b2, red)). (D) Thermal stabilization kinetics between TYMS (red) and SAMHD1 (blue) from the in-cell ITTRCETSA experiments with thymidine. Data is presented as two biological replicates at 37°C (TYMS_37°C, SAMHD1_37°C) and 52°C (TYMS_52°C, SAMHD1_52°C), with focus on the incubation duration of 0 to 30 mins. (E) Summary of hits identified by: in-cell MS-ITDRCETSA (dotted lines); lysate MS-ITDRCETSA (solid lines); validated by WB-ITDRCETSA (grey dotted lines). Common hits from the in-cell and lysate MS-ITDRCETSA (yellow circle); metabolites (grey circle); known metabolite-protein interactions (grey solid lines).