FIGURE 6.

Hrd-1 is an E3-ubiquitin ligase for tomosyn-2. A, INS1 (832/13) cells were co-transfected with plasmid expressing tomosyn-2-V5 along with GFP, Hrd-1 (wild type), or Hrd-1-dominant negative (DN). After 24 h, cells were incubated in growth media containing 3 mm glucose for 16 h. Next day, cells were treated with 11 mm glucose in the presence of 50 μm MG132 for 2 h. Cells were harvested for total protein. Immunoprecipitation (IP) was performed using anti-V5 antibody. The abundance of tomosyn-2, Hrd-1, and ubiquitin was determined by using anti-V5, anti-Hrd-1, and anti-ubiquitin antibodies, respectively. The data are representative of three independent experiments. WB, Western blot. ns, nonspecific band. B, HEK293FT cells were co-transfected with plasmid expressing tomosyn-2-myc with negative control shRNA or shHrd-1. Cells were harvest for total protein at 24, 48, and 72 h post-transfection. The abundance of tomosyn-2 and actin was determined by Western blot by using anti-Myc and anti-actin antibody, respectively. Graph shows relative protein abundance of tomosyn-2 normalized to actin. The abundance of tomosyn-2 after 24 h was set to one. Values are means ± S.E. of N ≥4. *, p ≤ 0.05 for the tomosyn-2 protein abundance in cells treated with shHrd-1 versus control shRNA.