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

FIGURE 2:

FIGURE 2:

Grp170’s nucleotide exchange activity triggers NHK release from BiP. (A) Coomassie staining of purified C-terminally FLAG-tagged GFP (GFP-FLAG), N-terminally FLAG-tagged WT Grp170 (WT Grp170-FLAG), and G41L Grp170 (G41L Grp170-FLAG) (B) Top, the BiP–NHK-S (10 nM) complex was incubated without ATP or with ATP (0.1 μM) for 10 min along with the indicated recombinant protein (125 nM). NHK-S was then reprecipitated and subjected to SDS–PAGE, followed by immunoblotting with anti–S-tag and BiP antibodies. Bottom, the BiP band intensity was quantified with ImageJ (National Institutes of Health, Bethesda, MD). Data represent mean ± SD of at least four independent experiments. (C) Cells expressing NHK-HA and BiP-S were transfected with scrambled or Grp170 #1 siRNA, the resulting WCEs were incubated with S-protein–conjugated beads, and the isolated proteins were subjected to SDS–PAGE, followed by immunoblotting with anti–S-tag and HA antibodies. Input WCEs were also analyzed by immunoblotting with an anti-HA antibody.