EMBO/soda-nel huggingface dataset:
Bipolar-associated miR-499-5p controls neuroplasticity by downregulating the Cav1.2 subunit CACNB2 (2022)
Figure 3

Figure 3: Elevated levels of miR-499-5p in hippocampal neurons impair cell surface expression and activity of Cav1.2 channels.

A) Representative images of DIV 19 rat hippocampal neurons co-transfected with GFP (green channel) and Cav1.2-HA (red channel), together with either control or miR-499-5p mimics. After 12-13 days of expression, labeling with Anti-HA antibodies was performed in live conditions to identify cell surface Cav1.2 channels. Whole cells: scale bars = 20 μm; cell body and dendrite insets: scale bars= 5 μm.

B) Quantification of the area occupied by surface Cav1.2 fluorescence normalized to the total cell area (GFP fluorescence) from non-permeabilized neurons transfected as in A). Data are represented as scattered dot plots with bar, mean±S.D. (n=4 independent experiments; Paired two-sample t-Test *p=0.0318).

C) Representative calcium current traces from neurons transfected with the indicated miRNA mimics.

D) I/V curves (current density against voltage) of calcium peak currents from hippocampal neurons co-transfected with GFP (n=9 cells) and the Control (n=10 cells) or miR-499-5p mimic (n=10 cells); (p=0.0188 for miR-499-5p mimic vs. Control mimic between −10 and +30 mV, Unpaired two-sample t-Test). Data are represented on XY graph as mean±S.E.M.

E) Representative calcium current traces from neurons transfected with the indicated miRNA mimics and pMT2-Cacnb2.

F) I/V curves (current density against voltage) of calcium peak currents from hippocampal neurons co-transfected with GFP (n=11 cells) and pMT2-Cacnb2 with the Control (n=11 cells) or with miR-499-5p mimic (n=12 cells) (n.s. p=0.5379 for miR-499-5p mimic vs. Control mimic between −60 and +60 mV, Unpaired two-sample t-Test). Data are represented on XY graph as mean±S.E.M.
Bipolar-associated miR-499-5p controls neuroplasticity by downregulating the Cav1.2 subunit CACNB2 (2022)
Figure 5

Figure 5: miR-499-5p expression is increased in human BD patients.

miR-499-5p qPCR analysis of total RNA isolated from PBMCs of control (female= 26, male= 31), BD (female= 26, male= 37), or MDD (female= 18, male=24) subjects. Wilcoxon rank sum test after correction for age and antidepressant treatment (linear model of the form Fold Change ~ Group*Sex + Age + Antidepressant treatment). 2-way ANOVA with correction for sex, age and antidepressant treatment (linear model of the form Fold Change ~ Group*Sex + Age + Antidepressant treatment). Control vs. BD: ****p=8.89e-07; control vs. MDD: n.s. p=0.12674. Data are presented as violin plots with median, quartiles and data points.

miR-499-5p qPCR analysis of total RNA isolated from PBMCs of control (female= 8, male= 10) and SZ (female= 8, male= 15) subjects. 2-way ANOVA, Main effect Sex: p=0.9785; main effect Group: p= 0.0924; main effect Interaction: p= 0.9802. Tukey's HSD: p=0.0924, ns. Data are presented as violin plots with median, quartiles and data points.

miR-499-5p qPCR analysis of total RNA isolated from PBMCs of control (female= 26, male= 31), BD I (female= 9, male= 9), or BDI II (female= 12, male=16) subjects. 2-way ANOVA, Main effect Sex: p=0.174; main effect Group: ****p=4.07e-09; Interaction Group x Sex: p=0.311. Tukey's HSD: control vs. BDI: ****p=0.0000037. control vs. BDII:p=0.0000002. Data are presented as violin plots with median, quartiles and data points.

miR-499-5p qPCR analysis of total RNA isolated from PBMCs of control subjects (female= 26, male= 31), and BD subjects in different mood states (depressive (female= 7, male=11), euthymic (female= 7, male=4), hypomanic (female=2, male=6), manic (female=2, male=1), mixed (female=1, male=1). 2-way ANOVA, main effect Sex: p=0.260; main effect Group: p=1.26e-07; Interaction Group x Sex: p=0.059; Tukey's HSD: control vs. depressive ***p=0.0002621, control vs. euthymic ***p=0.0008533, control vs. hypomanic ****p=0.0000470. Data are presented as violin plots with median, quartiles and data points.

miR-499-5p qPCR analysis of total RNA isolated from PBMCs of healthy control subjects (-CMT, n=17) and healthy subjects with a history of childhood maltreatment (+CMT, n=17). (****p<0.0001, Mann-Whitney U test). CMT: Childhood maltreatment. Data are presented as violin plots with median, quartiles and data points.
Bipolar-associated miR-499-5p controls neuroplasticity by downregulating the Cav1.2 subunit CACNB2 (2022)
Figure 6

Figure 6: miR-499-5p expression is negatively correlated with GMV in healthy human subjects.

A, B) Association of miR-499-5p expression and GMV in HC (n=23): miR-499-5p levels were negatively correlated with a GMV cluster comprising parts of the left Wernicke language area (i.e. transverse temporal gyrus, the left parietal operculum and the left superior temporal gyrus) (in blue) at p<.05 cluster-level family wise error-corrected (FWE) for multiple comparisons after an initial threshold of p<.001 uncorrected (k=1090, x/y/z=-42/-30/15, t= 4.7, p=.045). C) Proposed model for the mechanism of miR-499-5p dysregulation in BD. Early life stress induces the expression of miR-499-5p in human PBMCs and rat hippocampus, which impairs dendritic development and Cav1.2 calcium channel cell surface expression and activity by inhibiting the expression of an auxiliary subunit of Cav1.2 calcium channels and risk gene for BD, CACNB2.

Expanded Figure Legends:
ADAP and SKAP55 deficiency suppresses PD-1 expression in CD8+ cytotoxic T lymphocytes for enhanced anti-tumor immunotherapy. (2015)
Figure 1

SKAP55 enhances PD-1 expression to decrease CD8+ CTL cytotoxicity

A OT-I CD8+ CTLs were conjugated with 10 nM OVA257-264-pulsed EL-4 cells for 30 min, fixed, and stained with anti-SKAP55 (red), anti-LFA-1 (green) and Hoechst (blue).

B, C WT and SKAP55−/− OT-I CD8+ CTLs were generated from WT or SKAP55−/− OT-I Tg mice, then incubated with 10 nM OVA257-264-pulsed EL4 targets for 4 h to assess the in vitro cytotoxicity at different Effector:Target ratios (B; mean of triplicates ± SD); surface expression and the mRNA levels of PD-1 (C). Graphs are representative of at least three independent experiments.

D WT and SKAP55−/− OT-I CD8+ CTLs (3 × 106) were injected into C57BL/6 mice, followed by injection of non-pulsed (CFSEhi) or 10 nM OVA257-264-pulsed (CFSElo) splenocytes (5 × 106) to measure in vivo cytotoxicity (mean ± SD, n = 3 mice per group). Representative data of three independent experiments.

E WT and SKAP55−/− OT-I CD8+ CTLs were pretreated with anti-PD-1 antibody or IgG control, followed by incubation with OVA257-264-pulsed EL4 cells to examine in vitro killing ability (mean of triplicates ± SD). Graphs are representative of three independent experiments.

F CD8+ CTLs were transfected with plasmids expressing SKAP55-GFP or EGFP, then treated with non-pulsed or 10 nM OVA257-264-pulsed EL4 cells to examine surface PD-1 expression (left panel) or the in vitro killing assay (mean of triplicates ± SD) (right panel).

Data information: Statistical significance was determined with unpaired two-tailed Student's t-test. Graphs are representative of three independent experiments.
ADAP and SKAP55 deficiency suppresses PD-1 expression in CD8+ cytotoxic T lymphocytes for enhanced anti-tumor immunotherapy. (2015)
Figure 2

ADAP-deficient CD8+ CTLs reduce PD-1 expression and enhance cytotoxicity

A OT-I CD8+ CTLs were conjugated with CFSE-labeled OVA257-264-pulsed or non-pulsed EL-4 cells for 30 min, fixed, and stained with anti-ADAP (red).

B OT-I CD8+ CTLs were transfected with plasmids expressing ADAP or GFP, then stimulated with 10 nM OVA257-264-pulsed EL-4 cells to detect surface PD-1 expression. Representative of three independent experiments.

C, D WT and ADAP−/− OT-I CD8+ CTLs were stimulated with 10 nM OVA257-264-pulsed or unpulsed EL-4 cells for 4 h to examine surface expression and the mRNA levels of PD-1 (C), in vitro cytotoxicity at different Effector:Target ratios (D; mean of triplicates ± SD). Graphs are representative of at least three independent experiments.

E WT and ADAP−/− OT-I CD8+ CTLs were pretreated with 10 μg/ml anti-PD-1 antibody or IgG control, followed by incubation with 10 nM OVA257-264-pulsed EL4 cells to examine in vitro killing ability (mean of triplicates ± SD).

F Naïve CD8+ T cells were isolated from WT, SKAP55−/−, ADAP−/−, and SKAP55−/−ADAP−/− splenocytes, then stimulated with plate-bound anti-CD3/CD28 for 12 h to assess PD-1 mRNA levels, and for 48 h to check surface PD-1 expression (mean of triplicates ± SD).

Data information: Statistical significance was determined with unpaired two-tailed Student's t-test. Graphs are representative of three independent experiments.
ADAP and SKAP55 deficiency suppresses PD-1 expression in CD8+ cytotoxic T lymphocytes for enhanced anti-tumor immunotherapy. (2015)
Figure 3

The ADAP-SKAP55 module is dependent on NFATc1, Ca2+, and Fyn to control PD1 expression

A–C WT, ADAP−/−, or SKAP55−/− OT-I CD8+ CTLs were stimulated with OVA257-264-pulsed or unpulsed EL4 cells to detect the levels of NFATc1 expression, activation and nuclear entry by immunoblotting (A) or immunostaining (B). Alternatively, nuclear extracts from these CTLs were incubated with the N1 DNA probes containing NFAT-binding sites for an EMSA. The unlabeled competitor probe (Comp. N1) and the mutant probe that contains the same sequence except for carrying a mutated NFAT-binding site (Comp. mutN1) were included as controls (C).

D WT, ADAP−/−, or SKAP55−/− OT-I CD8+ CTLs were pretreated with DMSO or 5 μM CsA, then stimulated with OVA257-264-pulsed EL-4 cells for 4 h to examine surface PD-1 expression (mean of triplicates ± SD).

E WT, ADAP−/−, or SKAP55−/− OT-I CTLs were pretreated with the DMSO control, 10 μM PP2 or 75 μM 2APB, then stimulated with OVA257-264-pulsed EL-4 cells for 4 h to examine surface PD-1 expression (mean of triplicates ± SD).

F GFP, ADAP, or SKAP55 was overexpressed with pGL3-NFAT Luciferase reporter plasmid into Jurkat cells. Cells were stimulated with anti-CD3 and anti-CD28 in the presence or absence of PP2 or 2APB for 6 h, followed by measuring luciferase readings (mean of triplicates ± SD).

Data information: Statistical significance was determined with unpaired two-tailed Student's t-test. Graphs are representative of three independent experiments. Source data are available online for this figure.
ADAP and SKAP55 deficiency suppresses PD-1 expression in CD8+ cytotoxic T lymphocytes for enhanced anti-tumor immunotherapy. (2015)
Figure 4

SKAP55-deficient mice enhance DC-based vaccine for prevention of tumors in vivo

A WT and SKAP55 KO C57BL/6 mice were s.c. immunized on day −14 and day −7 by DCs that were prepulsed with B16F10 tumor lysates. On day 0, the immunized mice were i.v. inoculated with B16F10 cells. Number of lung tumors were counted 26 days after B16F10 challenge (n ≥ 5 mice).

B, C At day 0 (B, before tumor injection) or at day 26 (C, after tumor injection), surface PD-1 expression in CD44hiCD8+ or CD44hiCD4+ T cells, and the percentages of CD44hiCD8+ T cells or Foxp3+CD4+ cells were analyzed (mean ± SD, n ≥ 5 mice).

D, E The mRNA levels of PD-1, NFATc1, or the mRNA and protein levels of granzyme B in lung infiltrating CD8+ T cells were examined at day 26 (mean of triplicates ± SD, and the mRNA samples were prepared from at least 3 mice per group).

F CTLA-4 levels in the lung-infiltrated CD44hiCD8+ T cells or the percentages of CD4+Foxp3+ T cells in lungs, MLN, and spleen were measured at day 26 (mean ± SD, n ≥ 5 mice).

Data information: Statistical significance was determined with Mann–Whitney U-test. Data are representative of three independent experiments.
ADAP and SKAP55 deficiency suppresses PD-1 expression in CD8+ cytotoxic T lymphocytes for enhanced anti-tumor immunotherapy. (2015)
Figure 5

ADAP-deficient mice enhance DC-based vaccine for prevention of tumors in vivo

A The same procedures as in Fig4A were performed with WT and ADAP−/− C57BL/6 mice. HE staining of lung tissue was examined on day 26 (representative graph of 5 mice).

B, C PD-1 expression in CD44hiCD8+ or CD44hiCD4+ T cells (mean ± SD, n ≥ 5 mice per group) or granzyme B expression in CD44hiCD8+ T cells were examined from the lungs at day 26 (representative data of 3 mice per group).

D, E CTLA-4 levels in lung infiltrating CD44hiCD8+ T cells and the percentages of CD4+Foxp3+ T cells in the lungs or MLN were measured at day 26 (mean ± SD, n ≥ 5 mice per group).

Data information: Statistical significance was determined with Mann–Whitney U-test. Data are representative of three independent experiments.
ADAP and SKAP55 deficiency suppresses PD-1 expression in CD8+ cytotoxic T lymphocytes for enhanced anti-tumor immunotherapy. (2015)
Figure 6

SKAP55-deficient mice and SKAP55-deficient CTLs enhance anti-tumor therapy in vivo

A, B WT and SKAP55−/− mice were s.c. injected with MO5 melanoma cells followed by two injection at day 7 and day 14 of OVA257-264-pulsed DCs or the PBS control respectively (n ≥ 5). The growth of tumors under skin was measured every three days according to tumor diameter (mean ± SEM, n ≥ 5 mice per group) (A). Surface expression levels of PD-1 were checked on OVA257-264-specific Vα2high/Vβ5+ CD8+ effector cells at day 30 (mean ± SD, n ≥ 5 mice) (B).

C WT and SKAP55−/− mice were s.c. injected with MO5 melanoma cells followed by s.c. two injection of OVA257-264-pulsed DCs at day 7 and day 14. anti-PD-1 monoantibody or IgG controls were i.v. injected three times at day 7, day 10, and day 14 (50 μg/injection). The diameter and volume of tumors were measured every three days (mean ± SEM, n ≥ 5 mice).

D The WT recipient mice were s.c. injected with MO5 melanoma cells followed by injection of 10 nM OVA257-264-stimulated WT or SKAP55−/− CTLs at day 8 (n ≥ 8). The diameter and volume of tumors were measured every three days (mean ± SEM, n ≥ 8 mice).

Data information: Statistical significance was determined with Mann–Whitney U-test. Data are representative of two independent experiments.
CENP-C unwraps the human CENP-A nucleosome through the H2A C-terminal tail (2019)
Figure 1

Figure 1: CENP-A nucleosome has flexible DNA ends independently of the wrapped DNA sequence.

A. Graphs showing the relative abundance of undigested DNA as a function of time during digestion with micrococcal nuclease (MNase) for three types of DNA wrapped around the CENP-A nucleosome (red) and the H3 nucleosome (green). For α-satellite DNA with initial size of 171 bp, size ranges (141-171) corresponding to DNA lengths above NCP (nucleosome core particle) is presented. Data are presented as mean (SD) for each time point based on three independent experiments. Virtual gels from Bioanaylzer are in Figure EV1A.

B. CryoEM density map of the human CENP-A nucleosome, color-coded for histones and DNA.

C. CryoEM maps of the H3 nucleosome (PDB 6ESF) and the CENP-A nucleosome, colored based on local resolution.

D. Overlay of cryoEM maps of the H3 nucleosome (green; PDB 6ESF) and the CENP-A nucleosome (red). Note shorter and moved density for DNA on the CENP-A nucleosome indicated by the arrow.

E. Overlay of the N-terminal tail of CENP-A (red) and H3 (green; PDB 6ESF), illustrating shorter αN helix of CENP-A (obstructed by the presence of bulky CENP-AW47) and DNA moved by 4 Å.

F. The N-terminal tail of CENP-A (red) makes contacts with the DNA (cyan) at the entry/exit sites.
CENP-C unwraps the human CENP-A nucleosome through the H2A C-terminal tail (2019)
Figure 2

Figure 2: Both CENP-CCR and CENP-Cmotif bind specifically to the CENP-A nucleosome and CENP-CCR easily competes out CENP-Cmotif bound to CENP-A.

A. Schematic diagram of the full-length CENP-C protein, indicating parts involved in interactions with other proteins or homo-dimerization. Constructs used in this study are depicted below the diagram.

B. Native PAGE gel stained with Commassie blue showing complexes formed between CENP-A or H3 nucleosome and CENP-CCR. Lane 1: CENP-A nucleosome, Lanes 2-4: Increasing amounts of CENP-CCR are added to CENP-A nucleosome. Generation of a sharp band with slower mobility indicates formation of a specific CENP-A/CENP-CCR complex. Lane 5: H3 nucleosome. Lanes 6-8: Increasing amounts of CENP-CCR are added to H3 nucleosome. Smear on the gel indicates formation of non-specific CENP-A/CENP-CCR complexes.

C. Same experiment as in (B) using CENP-Cmotif. Lane 1. CENP-A nucleosome. Lanes 2-4. Increasing amounts of CENP-Cmotif are added to CENP-A nucleosome. Upon binding CENP-Cmotif, CENP-A nucleosome migrates slower through the gel. Note only modest change in mobility due to small size of CENP-Cmotif, comparing to CENP-CCR in (B). Lane 5. H3 nucleosome. Lanes 6-8. Increasing amounts of CENP-Cmotif are added to H3 nucleosome. Smear on the gel indicates formation of non-specific CENP-A/CENP-Cmotif complexes.

D. Native gel showing CENP-CCR competing out CENP-Cmotif bound to CENP-A nucleosome. Lane 1: CENP-A nucleosome, Lane 2: CENP-A/CENP-Cmotif complex. Lane 3-6: Increasing amounts of CENP-CCR are added to the pre-formed CENP-A/CENP-Cmotif complex. Formation of slower migrating bands indicates that longer CENP-CCR is replacing shorter CENP-Cmotif bound to the CENP-A nucleosome.

E. Native gel showing the inability of CENP-Cmotif to compete out CENP-CCR bound to CENP-A nucleosome. Lane 1: CENP-A nucleosome. Lane 2 and 6: CENP-CCR/CENP-A nucleosome complex. Lanes 3-5: Increasing amounts of CENP-Cmotif are added to the pre-formed CENP-A/CENP-CCR complex. Formation of smear at high amounts of CENP-Cmotif added, indicates that CENP-Cmotif, at high concentrations, non-specifically binds CENP-A/CENP-CCR complex rather than replacing bound CENP-CCR. Lane 7-9: Increasing amounts of CENP-CCR-short are added to the pre-formed CENP-A/CENP-CCR complex. Formation of bands with higher mobility indicates that smaller CENP-CCR-short is effectively replacing bigger CENP-CCR bound to CENP-A nucleosome.

Data information: For (B-E), 2.4 μM nucleosomes are used in all experiments.
CENP-C unwraps the human CENP-A nucleosome through the H2A C-terminal tail (2019)
Figure 3

Figure 3. Strong hydrophobic interactions are contributing specificity and high affinity between the CENP-A nucleosome and CENP-CCR.

A. Sequence alignment of CENP-CCR and CENP-Cmotif regions from different mammals. Conserved residues involved in CENP-A binding are highlighted in blue (electrostatic interactions) and pink (hydrophobic interactions). Residues identified in this study to contribute higher affinity of CENP-CCR comparing to CENP-Cmotif, are boxed.

B. CryoEM density map of the human CENP-A nucleosome, color-coded for histones, in complex with CENP-CCR (magenta). CENP-CCR binds CENP-A nucleosome through hydrophobic region (big box) and arginine anchor (small box). Interacting residues in each of the regions are shown in (C).

C. Ribbon diagram showing interactions of the CENP-CCR hydrophobic region (left) and CENP-CCR arginine anchor (right) with the CENP-A nucleosome.

D. Overlay of the CENP-A C-terminal tail before (gray) and after (red) binding of CENP-CCR.

E. Interactions between H3-GIEGGL and rat CENP-Cmotif as observed in PDB 4X23.

F. Schematic diagram of mutated CENP-CCR sequences used to test importance of CENP-CV532 and CENP-CV533 for generation of CENP-A/CENP-CCR complexes.

G. Native gels showing the impaired ability of mutated CENP-CCR to form complexes with the CENP-A nucleosome. The molar ratio of CENP-CCR/CENP-A nucleosomes is shown above each lane.
CENP-C unwraps the human CENP-A nucleosome through the H2A C-terminal tail (2019)
Figure 4

Figure 4. The C-terminus of H2A is destabilized in the CENP-A/CENP-CCR complex.

A. Graph showing the relative abundance of undigested DNA (145 bp) as a function of time during digestion with micrococcal nuclease (MN-ase) for the CENP-A nucleosome (red), the CENP-A/CENP-CCR complex (blue) and the CENP-A/CENP-Cmotif complex (black).

B. Overlay of three cryoEM maps (grey, blue and green) of the CENP-A nucleosome obtained by sorting on the DNA entry/exit site (Figure EV2F). Distance between most open (grey) and most closed (green) map is 2 Å. The entry/exit site is boxed and the corresponding model is shown on the right. Note that the density of the H2A C-terminus is well defined.

C. Overlay of three cryoEM maps (grey, blue and green) of the CENP-A/CENP-CCR complex obtained by sorting on the DNA entry/exit site (Figure EV4F). The distance between the most open (grey) and most closed (green) maps is 9 Å. Map assigned to CENP-CCR is shown in magenta. The entry/exit site is boxed and the corresponding model is shown on the right. Note the absence of a clearly defined density of the H2A C-tail (indicated by the dotted circle).

D. Same type of data as in (A) for the CENP-A nucleosome assembled with tailless H2A (H2A, 1-109) alone (pink) and in complex with CENP-CCR (light blue).

E. Schematic representation of the interplay between the N-terminus of H3 or CENP-A and the C-terminus of H2A in regulating flexibility of nucleosomal DNA wrap. DNA (cyan); longer H3 αN (green); shorter CENP-A αN (red); H2A C-terminus (yellow); CENP-C (magenta).

Data information: For (A, D) Data are presented as mean (SD) for each time point based on three independent experiments and virtual gels are available in Figure EV5.
The TBC1D31/praja2 complex controls primary ciliogenesis through PKA-directed OFD1 ubiquitylation (2021)
Figure 1

Figure 1. TBC1D31 binds and targets praja2 to the centrosome.

A. Co-immunoprecipitation of flag-praja2 and GFP-TBC1D31 from lysates of HEK293 cells. The immunoprecipitation (Ip) was performed using an anti-flag antibody or control IgG.

B, C. Same as in A, with the exception that cells expressing flag-praja2rm or praja2 deletion mutants (praja21-530, praja21-630 and praja2Δ530-630) were included in the analysis.

D. Lysates expressing flag-praja2 were subjected to pull down assay with GST and GST-TBC1D31940-970 polypeptides.

E. Co-immunoprecipitation of endogenous TBC1D31/praja2/PKAc complex from cell lysates.

F. Staining of HEK293 cells with anti-TBC1D31, anti-γ-tubulin and anti-praja2 antibodies. Nuclei were stained with DRAQ5 (blue). Where indicated, cells were transfected with GFP-TBC1D31. Arrows indicate the pool of praja2 colocalizing with TBC1D31 staining at the centrosome.

G. Cells transfected with control siRNA (siCNT) or siRNA targeting TBC1D31 (siTBC1D31) were stained for praja2, anti-γ-tubulin and DRAQ5.

H. Schematic picture of TBC1D31/praja2/PKA complex.
The TBC1D31/praja2 complex controls primary ciliogenesis through PKA-directed OFD1 ubiquitylation (2021)
Figure 2

Figure 2. Modelling TBC1D31/praja2 binding in vitro.

A. Lysates from HEK293 cells expressing GFP-TBC1D31 were subjected to pull down assay with GST and GST-praja2531-631 polypeptides.

B. MST signal (normalized fluorescence) of P1 (red curve), P2 (green curve) and P3 (cyan curve) plotted against TBC1D31, at increasing concentrations of peptides. The threading modelled structure of the overlapping binding segment of praja2 (praja2550-570) is shown.

C. Co-immunoprecipitation of GFP-TBC1D31 and flag-praja2 ring mutant (flag-praja2rm) or praja2Δ550-570.

D. Threading modelled structure of TBC1D31, with a zoom of its C-terminus. Mutated residues are highlighted in stick colored by atom type.

E. MD derived binding mode of praja2530-570 (green cartoon) to the C-terminal region of TBC1D31 (red cartoon).

F. MST signal of P1 plotted against increasing concentrations of TBC1D31 peptides: wild-type (red curve), R948A-R951A (violet curve) and R957-R959D-H960A (orange curve).