PMC

Linker DNA length distribution predicted for simulated dinucleosome sequences with 15 bp periodic linker length (A–E) and uniform linker length (F). (A) True linker length distribution used for simulation of dinucleosome sequences. (B, C) Recovered linker length distribution under the kernel and mixture methods, respectively, for 2,000 simulated dinucleosome sequences. (D, E) Corresponding results for a subset of the simulated dinucleosomes comprising only 300 sequences. (F) Recovered linker lengths using the kernel method for 2000 simulated dinucleosome sequences under the same model as in (B )but with a uniform linker length distribution on [1,…120].

(A) and (B) HEK-293 cells were co-transfected with Myc-Smad3 (Linker), Myc-Smad3 (Full Length), p300–HA and constitutively active TGF-β type I receptor TβRI (T204D) for TGF-β induction. Cell lysates were immunoprecipitated (IP) by HA antibody, followed by immunoblotting with an antibody against the Myc epitope. Lysate controls are shown for Myc-Smad3 (Linker) and Myc-Smad3 (Full Length) expression levels by Myc immunoblotting, and p300–HA levels by HA immunoblotting. (C) HEK-293 cells were co-transfected with GAL4–Smad3 (Linker), GAL4–Smad3 (Full Length), p300–HA and TβRI (T204D) for TGF-β induction. Cell lysates were immunoprecipitated by HA antibody, followed by immunoblotting with an antibody against the Smad3 linker region. GAL4–Smad3 (Linker) and GAL4–Smad3 (Full Length) expression levels were examined by the antibody against Smad3 linker region. p300–HA levels were examined by HA immunoblotting.

The linker region of the ISS exhibits variable conformations. (A) Representative 2D class averages of the chimeric ISS full-length protein. The linker region is clearly visible and highlighted with a white circle in the right panel. (B) Brief flowchart of EM data processing for the ISS linker region. A characteristic class featured with visible TM region and linker region (highlighted in red box) was obtained from 3D classifications (details in Materials and Methods and Fig. S7). Using 3D classification with this map as reference, the linker region was classified into variable conformations, with two major orientations, named left and right linkers (bottom-left panel). For independent validation of variable states of the linker region, a 3D classification by skipping alignment with a local mask in the linker region was performed and classes of the linker region in different orientations (left and right linker) were obtained (bottom-right panel). (C) Structural comparisons for linker regions of the ISS maps. Structural presentation and overlay comparison for left (colored in yellow) and right linker (colored in cyan) in whole maps of the ISS (top panel). Structural presentation and overlay comparison for left (colored in marine) and right linker (colored in pink) in maps of the ISS with a local mask in the linker regions (bottom panel). Notably, a few of features of TM region can be seen in the whole maps of the ISS.

(A) Rationale of the DD-technology: Fusion of PTEN with a modified human FKBP variant harboring the F36V and L106P point mutations (FKBP*) leads to degradation of the fusion protein. Presence of the FKBP* ligand Shld1 confers stabilisation of FKBP*-PTEN and results in inhibition of PI3K/Akt signaling. (B) The fusion protein FKBP*-PTEN shows no PTEN activity. PTEN-deficient U87MG cells were transiently co-transfected with FKBP*-PTEN and AKT-GFP. After one day, cells were treated with Shld1 for 12 hours at 1μM before cell lysis and samples analyses by western blotting using indicated antibodies. (C) Consequence of linker optimization on PTEN activity of the FKBP*-PTEN fusion protein. Schematic representation of linker optimization. FKBP*-linker-PTEN variants with different linker regions (A-E) were generated. Linker A—short flexible linker; Linker B—a helix forming linker. Linker C—long flexible linker. Linker D—long flexible linker containing the PWR motif. Linker E—the original linker present in the enzymatic active GFP-PTEN. (D) Analyses of the effect of different linkers on PTEN activity in FKBP*-PTEN fusion variants in U87MG cells. Constructs expressing FKBP*-PTEN variants or GFP-PTEN were co-transfected with AKT-GFP into a PTEN null U87MG cells. Stabilization of FKBP*-PTEN or GFP-PTEN in presence or absence of Shld1 was monitored by PTEN antibody. The phosphorylation level of AKT-GFP at Ser473 served as a fast and effective experimental protocol to control for PTEN activity towards PIP3-dependent signaling in transfected cells, only. Only constructs 4 and 5, which contain proline in the linker sequence, showed moderate PTEN activity towards PI3K signaling.