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. 2015 Apr 29;16(6):709–718. doi: 10.15252/embr.201440006

Figure 2.

Figure 2

Starvation induces DENND3 phosphorylation via ULK1/2
  • A Lysates from HEK-293T cells transfected with Flag-DENND3 wild-type (WT), S554A or S572A mutant were incubated with protein G beads alone or protein G beads coupled to anti-Flag monoclonal antibody (IP-Flag). Proteins bound specifically to the beads were processed for Western blot with anti-Flag and anti-pan-14-3-3 antibodies. An aliquot of the cell lysate (starting material, SM) equal to 10% of that added to the beads was analyzed in parallel (n = 3 repeats).
  • B Schematic diagram of DENND3 with a N-terminal DENN domain, a C-terminal WD40 domain, and a presumably weakly structured linker region (white box). The aligned sequences are from a region of the linker and indicate the high degree of conservation across various vertebrate species. S554 and S572 (position based on the mouse sequence) are indicated.
  • C HeLa cells were left unstarved (0 min) or were starved with EBSS for the indicated time periods. Lysates were immunoblotted with antibodies recognizing the indicated proteins.
  • D Relative DENND3 phosphorylation at serine 554 and serine 572 was determined from 3 experiments as in (C). Points represent mean ± SEM. Statistical analysis employed one-way ANOVA followed by Dunnett's post-test. ***P < 0.001, *P < 0.05.
  • E HeLa cells were processed as in (C), followed by quantitative real-time PCR (n = 4 repeats). Bars represent mean ± SEM. Statistical analysis employed one-way ANOVA followed by Dunnett's post-test. NS = not significant.
  • F Purified GST-DENND3 (538–973) was subjected to in vitro phosphorylation by purified HA-ULKs (n = 3 repeats). Note that the WT ULK1 and ULK2 run higher on SDS–PAGE than the kinase-inactive forms, due to autophosphorylation, and a shift of the phosphorylated GST-DENND3 can also be seen on a Ponceau-stained transfer.
  • G HeLa cells were transfected with control siRNA or siRNA targeting ULK1 or ULK2 and were subsequently left unstarved or starved with EBSS for 10 min. Lysates were immunoblotted with the indicated antibodies (n = 3 repeats).
  • H For validating the ULK2 siRNA knockdown efficiency without workable antibody, HeLa cells were transfected with HA-tagged ULK2 with or without siRNA for ULK2 and lysates were immunoblotted with the indicated antibodies (n = 3 repeats).
  • I, J Relative DENND3 phosphorylation at S572 (H) and S554 (I) was determined from three repeats as in (F). Bars represent mean ± SEM. Statistical analysis employed one-way ANOVA followed by Tukey's post-test. ***P < 0.001, **P < 0.01. *P < 0.05.
  • K HeLa cells were processed as in (G), followed by qPCR (n = 4 repeats). Bars represent mean ± SEM. Statistical analysis employed one-way ANOVA followed by Tukey's post-test. NS = not significant.