Skip to main content
. 2016 May 15;27(10):1650–1662. doi: 10.1091/mbc.E16-01-0033

FIGURE 4:

FIGURE 4:

Depleting Grp170 traps the NHK-BiP complex on Sel1L. (A) Cells expressing NHK-HA were transfected with scrambled siRNA or Grp170 siRNA #1, treated with cycloheximide and MG132 for 4 h, and harvested. After lysis, the resulting WCEs were subjected to immunoprecipitation using an anti-HA antibody. The resulting precipitated proteins were analyzed with SDS–PAGE, followed by immunoblotting with anti–FLAG-tag and S-tag antibodies. Input WCEs were also analyzed with an anti-Sel1L antibody. (B) Cells expressing NHK-FLAG and BiP-S were transfected with scrambled siRNA or Grp170 siRNA #1, treated with cycloheximide and MG132 for 4 h, and harvested. The resulting WCEs were subjected to immunoprecipitation using FLAG antibody–conjugated beads. The bound proteins were eluted with FLAG peptide and separated with SDS–PAGE, followed by immunoblotting with the indicated antibodies. Input WCEs were also analyzed. In addition, the eluted protein samples were further incubated with the S-protein–conjugated beads to pull down BiP-S, and the isolated proteins were analyzed by immunoblotting with the indicated antibodies.