Fig 1.
Expression of AKT nanobodies in WK6 E. coli.
Western blot detection of AKT Nbs in a crude periplasmatic extract on a 15% SDS gel. An anti-HA Ab was used to detect the Nbs. The vast majority of the Nbs had comparable and high expression yields. The expression of AKT1 Nb14 and Nb15, AKT1-E17K Nbs5-8, AKT2 Nb10 and AKT3 Nb1 was low (denoted by an arrow) whereas only AKT1 Nb17 and AKT3 Nb11 could not be detected. Uncropped blots are available in S1 Raw images.
Fig 2.
ELISA screening of AKT pleckstrin homology domain nanobodies.
The mean and 95% confidence interval (CI) of OD405 values are shown. Pleckstrin Homology domains were coated in wells of a 96 multiwell plate at 1μg/ml and incubated with the AKT Nbs (20μl from a crude periplasmatic extract). The EGFP Nb was used for background correction. A Nb was considered to interact with a PH domain when the OD405 fold change (normalized to the OD405 of the EGFP Nb for the same PH domain) was at least three (denoted by a dashed line). Nbs, which did not meet this criterion for any PH domain are not shown on this Figure and were not included in further analysis. ELISA data for the complete Nb sets are available in S2 Fig.
Fig 3.
Co-IP of AKT isoforms using AKT1-, AKT1E17K-, AKT2- and AKT3-Nbs.
Immunoprecipitation of endogenous AKT1, AKT2 or AKT3 from MDA-MB-231 cells with recombinantly produced HA-tagged Nbs. Representative for three repeated experiments. For the AKT isoforms a pair of panels is shown for each isoform with a short exposure time and a long exposure time. The short exposure panel contains no saturated pixels. WCL = whole cell lysate, EGFP Nb = negative control of anti-HA-agarose incubated with the EGFP Nb and MDA-MB-231 lysate and LC = Light-Chain. Nbs were blotted using an anti-HA antibody, AKT1, AKT2 and AKT3 were detected using the C73H10, D6G4 and 62A8 isoform-specific Ab clones respectively. All nanobodies (Nb-HA) were efficiently expressed in E. coli. Uncropped blots are available in S1 Raw images.
Fig 4.
(A) Illustration of the AKT fragments used for epitope mapping. (B) ELISA: Mean and 95% CI of normalized OD plotted for full-length AKT2 (FL-AKT2), the AKT2 PH domain (AKT2PH), a fragment consisting of the flexible linker, kinase domain and regulatory domain (AKT2ΔPH) and the regulatory domain (AKT2REG). Target proteins were coated in wells of a 96 multiwell plate in quadruplicate for each AKT2 Nb and the EGFP Nb (negative control). All measured OD’s were normalized for the negative control. A Nb was considered to be an interactor when the average normalized OD was at least three times that of the negative control for the same AKT2 fragment (dashed line).
Fig 5.
AKT Nanobodies interfere with AKT-PH—PIP3 interaction.
(A) Western Blot of pull-down experiments using PIP3—coated beads and the AKT PH-domains. For each PH domain a positive control (CTRL) was included where the PH-domains were incubated with the beads without Nb. When the PH-domains were incubated with AKT1-E17K Nb7 or AKT2 Nb9 before PIP3-coated beads were added we observe a reduction in the signal for AKT1 PH- and AKT1-E17K PH- or AKT2 PH-domain respectively. Representative for three repeated experiments. *p≤0.05, **p≤0.01 (paired t-test). The AKT1-, AKT1-E17K-–and AKT2-PH-domain were detected using an Ab specific for AKT1 (C73H10) and AKT2 (D6G4) respectively. Uncropped blots are available in S1 Raw images. (B) ImageJ quantification of western blots. All signal intensities were normalized for the appropriate control (CTRL). The mean (bar) and SD (whiskers) are shown.
Fig 6.
Co-IP of endogenous AKT from MDA-MB-231 cells with Nbs transiently expressed as intrabodies.
(A) Co-IP of AKT and Nbs. WCL = whole cell lysate from EGFP Nb transfected cells, The negative control were cells with transient expression of the EGFP Nb. LC = Light Chain. (B) Nb expression in crude lysate. 10μg crude lysate from transfected cells was analysed through SDS-PAGE and western blotting. GAPDH signal was used as loading control. AKT was detected using a pan-AKT antibody (C67E7), Nbs were detected using an anti-V5-antibody. Uncropped blots are available in S1 Raw images.
Table 1.
Summary of characteristics for relevant AKT nanobodies.