Fig. 2: USP39 controls the stability and activity of the PDH complex.
From: USP39 regulates pyruvate handling in non-small cell lung cancer
A LC–MS/MS analysis of immunoconjugates pulled down with USP39 antibody. Number of peptides and coverage of USP39, DLAT, and PDHA. B Western blot analysis of immunoconjugates from (A). C Cellular fractionation of NCI-H1975 cells. Lamin B1, TOMM40 and Tubulin serve as markers for nucleic, membrane, and cytosolic fractions respectively. D Protein levels of PDH complex components after 48 h of USP39 siRNA-mediated silencing with quantification (n = 3). E Protein levels of PDHA after 48 h of USP39 siRNA-mediated silencing and treatment with 5 µM of MG132 for 4 h (n = 3). F Enzymatic activity of the PDH complex (n = 3) after 48 h of USP39 knockdown. G mRNA expression levels of PDHA and DLAT upon 48 h of USP39 silencing with siRNAs (n = 3). H In vitro deubiquitination assay showing the cleavage of Lys48 and Lys63 U1-7 polyubiquitin chains after incubation with recombinant USP39 for 16 h. I Ubiquitination level of PDHA in USP39-overexpressing cells and after treatment with MG132 for 4 h (n = 3). Quantification of ubiquitination levels were normalized to total PDHA levels and to the control. J Lys63-ubiquitination level of PDHA in USP39-knockdown cells and after treatment with MG132 for 4 h (n = 3). Quantification of Lys63-ubiquitination levels were normalized to total PDHA levels and to the control. K Protein level of PDHA after 72 h of USP39 overexpression with quantification (n = 3). Error bars ± SD. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
