Fig 2: Increased level of 14-3-3t protein in involved compared with uninvolved psoriatic skin. Protein extracts prepared from keratome biopsies taken from involved and uninvolved psoriatic skin were analysed with Western blot technique. (A) Shows the membrane probed with an antibody detecting 14-3-3t. ß-actin was added to demonstrate equal protein loading. Pt. 1–4 is randomly chosen between the nine patients totally included. (B) Shows the protein level of all seven 14-3-3 isoforms. Band intensity was quantified using Kodak 1D Image Analysis Software. The protein level was normalized to ß-actin and uninvolved biopsies were indexed to one. Bars indicate mean ± SD. *P<0.05.

Fig 3: Binding between 14 and 3-3 proteins and TFE3 fusion proteins is reduced in the UOK109 and UOK120 cells. (a) 786-O cells were treated with PP242 (4 µM) or vehicle control (Con) for 24 h followed by immunoblot analysis of 14–3-3 proteins. GAPDH was used as an internal control. (b) HK-2 cells were treated with PP242 (4 µM) or vehicle control (Con) for 24 h followed by immunoblot analysis of 14–3-3 proteins. ß-Tubulin was used as an internal reference. (c, d) UOK109 and UOK120 cells were treated with PP242 (4 µM) or vehicle control (Con) for 24 h followed by immunoblot analysis of 14–3-3 ?, 14–3-3 ?, p-S6, S6 and mTOR proteins. (e, f) HK-2 cells were treated with Torin1 (4 nM) or vehicle control (Con) for 24 h followed by immunoblot analysis of 14–3-3 ?, phosphor-S6 (p-S6), S6 and mTOR proteins. (g) The binding between TFE3 and 14–3-3 ? was detected by co-immunoprecipitation (CO-IP). An irrelevant IgG was used as negative control

Fig 4: 14-3-3 mRNA expression in psoriasis (A), basal cell carcinoma (B), atopic dermatitis (C) and contact dermatitis (D). mRNA expression was analysed in paired punch biopsies from involved and uninvolved skin by quantitative reverse transcription-polymerase chain reaction. mRNA levels were normalized to the housekeeping gene RPLP0 and uninvolved biopsies were indexed to one. Bars indicate mean ± SD. *P<0.05. (n=9 for psoriasis, n=3 for BCC, atopic dermatitis and contact dermatitis).

Fig 5: GPIba and platelet function are unperturbed by 14-3-3? deficiency.(a) Hirudin-anticoagulated blood from 14-3-3?-wt and 14-3-3?-deficient (14-3-3?-null) mice was perfused through collagen-coated (250 µg ml-1 Type I) microslides at 1,800 s-1 for 5 min. (i) DIC images are taken from 1 representative of four independent experiments (scale bar, 20 µm). (ii) Thrombi surface coverage was quantified using ImageJ software (4–5 fields analysed per flow). The histogram depicts the mean±s.e.m. (n=5; analysed using two-way ANOVA with Bonferroni’s post hoc testing). (b) VWF binding was measured as described under ‘Methods’. The graph represents one of three independent experiments. (c) Aggregation of washed mouse platelets in the presence of human VWF (10 µg ml-1) and botrocetin (10 µg ml-1), with stirring. The graph depicts aggregation traces from one representative of three independent experiments. (d) Comparative aggregation of washed 14-3-3?-wt and 14-3-3?-null platelets in response to CRP or ADP ((i) ADP 1 µM; (ii) CRP 20 ng ml-1). Results are taken from one representative experiment. A histogram depicting the mean±s.e.m. (n=3; analysed using a two-way ANOVA with Bonferroni’s post hoc testing), is presented in Supplementary Fig. 2b. (e,f) Diluted whole blood samples from 14-3-3?-wt (black bars) or 14-3-3?-null (white bars) mice were incubated with PE-JON/A (e) or FITC-anti P-selectin antibody (f), as described under ‘Methods’, to examine integrin aIIbß3 and degranulation of platelet a-granules, respectively, and analysed by flow cytometry, following incubation with the indicated agonist/concentration for 15 min. Results depict the % of gated platelets positive for antibody binding and are expressed as the mean±s.e.m. ((e) PAR4P: n=10; CRP: n=3; ADP: n=4; (f) PAR4P: n=10; CRP: n=5), where NSP>0.05. Note: no significant difference was observed in the geometric mean of fluorescence intensity for the same experiments. (g–i) Resting whole cell lysates were prepared from 14-3-3?-wt (wt) and 14-3-3?-null (null) washed platelets. (g) Expression levels of 14-3-3 isoforms were compared using SDS–PAGE and immunoblot analysis, as described under ‘Methods’, with immunoblots probed with the indicated 14-3-3 isoform-selective or pan-14-3-3 antibodies. These studies confirm deletion of 14-3-3? protein in the 14-3-3?-null mice, with some upregulation of 14-3-3?. (h,i) Association of 14-3-3 proteins with the GPIba cytoplasmic tail in the absence of 14-3-3?—GPIba was immunoprecipitated from 14-3-3?-wt and 14-3-3?-null platelet lysates as described under ‘Methods’, and analysed via immunoblotting, using (h) anti-14-3-3? or (i) anti-pan-14-3-3. Immunoblots are taken from one representative of three independent experiments.