Journal: Journal of Cachexia, Sarcopenia and Muscle
Article Title: A muscle‐specific MuRF1‐E2 network requires stabilization of MuRF1‐E2 complexes by telethonin, a newly identified substrate) A muscle‐specific MuRF1‐E2 network requires stabilization of MuRF1‐E2 complexes by telethonin, a newly identified substrate
Figure Lengend Snippet: E2E1, E2G1, E2J1, E2J2, and E2L3 interact with MuRF1 in the presence of telethonin (A) Telethonin does not interact with E2s. Y2H experiments were performed using telethonin as a bait to confirm that this protein cannot directly interact with the E2 enzymes used in this work. The empty vector and the vector containing the LT construct were used as negative controls against telethonin to estimate potential background level. Signals above ‘empty’ and ‘LT’ lanes were considered as positive. Colonies were plated on selective medium [ ‐LTH + Aureo + 3‐AT ] (Experimental section) and monitored during 21 days. LT, Large‐T antigen; Tele, telethonin. (B) Telethonin expression level in yeast varies a ccording to methionine (Met) concentration in the medium. BD‐Tele, fusion protein between the binding domain of GAL4 and telethonin; Tele, telethonin. (C) Densitometry analysis from the immunoblot presented in (B). (D) Yeast three‐hybrid (Y3H) experiments revealed E2E1, E2G1, E2J1, E2J2, and E2L3 as MuRF1 partners in the presence of telethonin. E2‐expressing yeasts were mated against strains expressing either MuRF1 alone or MuRF1 and telethonin. Colonies were plated on selective medium [ ‐LTH + Aureo + 3‐AT ] containing 134 mM Met. Results were observed at day 6. Three to four independent transformation experiments were performed and 11 to 32 colonies were analyzed for each E2.
Article Snippet: Please refer to Table for other E2 enzymes nomenclature. cDNAs were cloned in yeast two‐hybrid vectors pGADT7, producing a fusion protein with the activation domain (AD) of GAL4 transcription factor, or in pGBKT7 and pBridge vectors, producing a fusion protein with the binding domain (BD) of GAL4 (Clontech).
Techniques: Plasmid Preparation, Construct, Expressing, Concentration Assay, Binding Assay, Transformation Assay