Fig. 1: MED15 T603 testified as a phosphorylation site involved in TGFβ-inhibited cell growth.
From: A phosphorylation switch in the Mediator MED15 controls cellular senescence and cognitive decline
a Endogenous co-IP assay using HA-beads to pull down HA-MED15 from 293T cells. The HA tag was knocked into the N-terminus of MED15. b Tandem mass spectrometry analysis of the MED15 peptides modified by phosphorylation of the T603 residue. c Posttranslational modification analysis of MED15 by PhosphoSitePlus database. d Sequence alignment of MED15 proteins across different species. The blue bold T indicates the MED15 T603 site. e Endogenous MED15 T603A mutant genotyping via PCR and Sanger sequencing in HaCaT cells. f CCK-8 assays were used to assess the proliferation rates of WT and T603A mutant HaCaT cells treated without or with 5 ng/mL TGFβ. The values are presented as means ± SD. ***P < 0.001. g Statistical analysis of the cell cycle distribution of WT and T603A mutant HaCaT cells treated without or with 5 ng/mL TGFβ. The values are presented as means ± SD. **P < 0.01. h qRT-PCR was used to determine the mRNA levels of the TβR genes in MED15 T603A cells and control cells treated without or with 2 ng/mL TGFβ. The values are presented as means ± SD. **P < 0.01 and ***P < 0.001. i Immunoblot analysis with the indicated antibodies in WT and MED15 T603A mutant HaCaT cells treated without or with 2 ng/mL TGFβ. β-Actin and GAPDH were blotted as loading controls.
