Figure 4.

Function of GAPDH arginines 197 and 200 in TRAF2 polyubiquitination. A, GAPDH knock down efficiency. HEK293T cells were transfected with the shRNA plasmid psi-LVRU6GP targeting the GAPDH 3′-UTR mRNA. Stable cell lines were created using puromycin selection. Tubulin was used to normalize GAPDH abundance. Asterisks indicate protein abundance significantly different from that of the control (n = 3, ANOVA). B, GAPDH R197/Arg²⁰⁰ mutants neither interact with nor activate TRAF2 polyubiquitination in HEK293T cells. GAPDH stable cell line was co-transfected with FLAG-TRAF2, HA-ubiquitin together with either Myc-GAPDH WT or the indicated GAPDH mutants. After 48 h, cell lysates were immunoprecipitated (IP) with anti-FLAG antibody, followed by immunoblotting with anti-ubiquitin or anti-Myc antibody. The abundance of endogenous GAPDH and transfected forms of GADPH-Myc is also shown. Asterisks used in quantification panels indicate significantly different TRAF2-Ub signal intensity as compared with the TNF-α control (n = 3, ANOVA). Endo., endogenous; Exo., exogenous; Ub, ubiquitin.