Fig 2: Unspecific detection of PKC with the Thr(P)308-Akt antibody in neuronal cells. A, cell lysates obtained from nontreated or TPA-treated 21 DIV cortical neurons were analyzed by Western blotting. Thr(P)308-Akt antibody detection identified phosphorylated Akt, as well as a slower migrating Thr(P)308-immunoreactive band of approximately 70 kDa. This band was sensitive to PKC inhibition with long term TPA treatment for 24 h. Thr(P)308-Akt was not affected. B, the three additional peaks in the cIEF profile recognized by Thr308 but not Ser473 (pI 5.68, 5.76, and 5.85) were not detected after TPA treatment.

Fig 3: Palmitoylation was required for SCP1-mediated AKT inhibition(A) Palmitoylation was required for SCP1 to dephosphrylate AKT Ser473. HEK293T cells were transfected with WT-SCP1, DN-SCP1, and 2S-SCP1 for 24 h and treated with DMSO or 2-bromopalmitate (2BP; 10 μM) for 6 h. The phosphorylations of p-Ser473-AKT and p-Thr450-AKT were detected using western blotting. (B) Palmitoylation of SCP1 at C44 and C45 was required for its suppression of cell proliferation. Values represent mean ± SD (n = 3) (C) Palmitoylation inhibition increased the phosphorylation levels of endogenous AKT Ser473. HEK293T cells and H1299 cells were treated with DMSO or 2BP (10 μM) for 6 h. The phosphorylation of p-Ser473-AKT was detected using western blotting. (D) Depalmitoylation blocked the interaction between SCP1 and AKT. HEK293T cells were transfected with Myr-AKT-HA and FLAG-SCP1 or FLAG-SCP1-2S for 24 h and treated with DMSO or 2BP (10 μM) for 6 h. The interaction between AKT and SCP1 or SCP1-2S was detected using western blotting. (E) SCP1 blocked HUVEC migration in a palmitoylation-dependent manner. HUVECs were transfected with vector, WT-SCP1, DN-SCP1, and 2S-SCP1, respectively. Cell migration was detected using a wound healing assay. (F) SCP1 blocked HUVEC tube formation in a palmitoylation-dependent manner. HUVECs were transfected with vector, WT-SCP1, DN-SCP1, and 2S-SCP1, respectively. Tube formation was detected using a tube formation assay. (G) The working model for SCP1 palmitoylation and cell membrane localization is shown.DOI: http://dx.doi.org/10.7554/eLife.22058.015

Fig 4: Context-dependent Akt phosphorylation signatures in cortical neurons. 21-DIV cortical neurons were stimulated with BDNF, EGF, or insulin for 15 min. In parallel, cortical neurons obtained from PTENfl/fl mice were infected at 12 DIV with Cre virus and cultured until 21 DIV. All cell lysates were analyzed by cIEF with Ser(P)473-Akt antibody (A) and Thr(P)308-Akt antibody (B). Although Akt peak distributions in response to growth factor treatment are largely identical, PTEN loss induces a strong increase in the most acidic Akt1 phosphorylated peaks.

Fig 5: Dynamics of Akt phosphorylation in response to insulin in N1E-115 cells. A, N1E-115 cells were starved for 48 h before insulin stimulation for different times (1–15 min). Cell lysates were analyzed by Western blotting using indicated antibodies. B and C, NIE-115 cell lysates were analyzed in parallel by cIEF using Ser(P)473-Akt antibody (B) and Thr(P)308-Akt antibody (C). D and E, the area under each peak was quantified for Ser(P)473-Akt (D) and the Thr(P)308-Akt antibody (E), demonstrating the phosphorylation dynamics in time of individual peaks.