Figure 1.

SHP is rapidly degraded by the ubiquitin–proteasome pathway. (A) Experimental outlines for in vivo experiments are shown in Supplemental Figure S1. Briefly, Ad-Flag-SHP or control Ad-empty (E) was injected via the tail vein and, 5 d later, mice were fed normal (−) or CA-supplemented (+) chow for 5 h and liver extracts were prepared. Flag-SHP and associated hepatic proteins affinity-purified on M2 agarose were visualized by silver staining. One band (arrow) was identified as psmd1 by mass spectrometry. (B) HepG2 cells transfected with a Flag-SHP expression plasmid were treated with proteasome inhibitors as indicated for 6 h and subjected to Western analyses. Duplicates are shown. (C) Hepa1c cells were transfected with pcDNA3-Flag-SHP with (+) or without (−) an HA-ubiquitin plasmid and then were treated with vehicle or MG132 for 3 h. Flag-SHP was immunoprecipitated from cell extracts with M2 antibody (IP) and ubiquitinated SHP in the immunoprecipitates was detected by Western analysis (WB). Heavy and light chains of IgG are indicated by asterisks. Positions of ubiquitinated SHP proteins are indicated by a dotted line. Flag-SHP levels in the input are shown in the bottom panel. (D) HepG2 cells infected with Ad-Flag-SHP were metabolically labeled for 30 min followed by chase for the indicated times. Flag-SHP was immunoprecipitated with M2 antibody and detected by autoradiography. (NS) Nonspecific band. Band intensities were measured by densitometry and the intensities relative to the 0-min time point were plotted. (E) HepG2 cells infected with Ad-Flag-SHP were treated with CHX (10 μg/mL) for the indicated times and Flag-SHP levels were detected by Western analyses. Band intensities were quantified as in D. SEM, n = 4. (F) HepG2 cells were treated with CHX for the indicated times and endogenous SHP was immunoprecipitated with SHP antibody or IgG control and detected by Western analyses. As a control, tubulin levels were also detected.