Fig 3. RLIM does not affect c-Myc protein degradation.
(A) and (B) 293T (A) and H1299 (B) cells were transfected with myc-c-Myc and increasing amount of HA-RLIM or HA-RLIMC596A plasmids as well as GFP plasmid. Ectopic c-Myc protein was detected by anti-myc antibody. GFP was used to monitor transfection efficiency. c-Myc and GFP levels were quantified using ImageJ software and the ratios of c-Myc to GFP are shown. (C) and (D) 293T (C) and H1299 (D) cells were transfected with scramble siRNAs or siRNAs against RLIM. Endogenous c-Myc protein was detected by anti-c-Myc antibody. c-Myc and actin levels were quantified using ImageJ software and the ratios of c-Myc to actin are shown. (E) and (F) 293T cells were transfected with HA-RLIM (E) plasmid or siRNA against endogenous RLIM (F). Cells were harvested at different time points after cycloheximide treatment and subjected to WB. Quantification of c-Myc protein level relative to actin are summarized from 3 independent experiments and shown in the right panels.