Secretion of wild type LGI1 co-expressed with LGI1 mutant proteins.

<p>Western blot of concentrated media of HEK293T cells co-transfected with GFP-tagged wild type (wt) LGI1 and one of the indicated Flag-tagged LGI1 constructs: wt, R407C (sec+), or I122K (sec-). Bands were revealed with an anti-LGI1 antibody. IgG gel loading control added to cell culture medium prior concentration is shown below. Vector, empty vector; MW, molecular weight markers.</p

Genetic features of study mutations.

<p>Genetic features of study mutations.</p

Percentage of cells with LGI1 (wild type or mutated) bound to either ADAM22 or ADAM23 receptor on the cell membrane.

<p>Percentage of cells with LGI1 (wild type or mutated) bound to either ADAM22 or ADAM23 receptor on the cell membrane.</p

LGI1 mutations impair interaction with ADAM22 and ADAM23.

<p>HEK293T cells were transiently co-transfected with (A) HA-ADAM22 or (B) HA-ADAM23 and LGI1 wild-type or mutants. Inputs (6.5% of total proteins) and HA-immunoprecipitations were separated in SDS-PAGE, transferred and immunoblotted using the indicated antibody. Co-immunoprecipitations showed that wild type LGI1 interacts with HA-ADAM22 (A) and HA-ADAM23 (B), while the interaction is abolished in the presence of T380A mutant. LGI1 mutants R407C, S473L and R474Q displayed a reduced interaction compared to the wild-type. Each of these experiments was performed twice with similar results. In the experiment shown in panel B, HA-ADAM23 failed to transfect HEK293T cells together with LGI1-S473L.</p

Proportion of cells with LGI1-ADAM22 complex on the outer membrane surface in the presence or absence of LE patient serum.

<p>Proportion of cells with LGI1-ADAM22 complex on the outer membrane surface in the presence or absence of LE patient serum.</p

Secretion test of wild type and mutated LGI1.

<p>Western blot of cell lysates (L) and concentrated media (M) of HEK293T cells transfected with (left panel) LGI1 wild type or sec+ mutant, c.1138A>G (T380A), c.1418C>T (S473L), and c.1421G>A (R474Q), expression constructs, or with (right panel) the LGI1 sec- mutant, c.365T>A (I122K), construct. Bands were revealed with an anti-LGI1 antibody. Ponceau S staining of the blots are shown below. Vector, empty vector; MW, molecular weight markers.</p

Gene expression profile of CAergic markers in differentiated SH-SY5Y and BE(2)-M17 cells.

<p>After 4 and 7 days of differentiation with TPA, RA and staurosporine, TH, AADC, VMAT2 and DβH mRNA levels were compared with their corresponding levels in undifferentiated cells using qRT-PCR. Expression was displayed on a Log₂ scale. Positive and negative values indicated up and downregulation of the genes with respect to control cells (undifferentiated cells), respectively. For each gene, differences between differentiated and undifferentiated cells were tested for significance using Student’s t-test. <i>(*P<0</i>.<i>05</i>, <i>**P<0</i>.<i>01</i>, <i>***P<0</i>.<i>001)</i>.</p

Cathecolamine contents detected in SH-SY5Y and BE(2)-M17 cell lines before and after differentiation for 7 days.

<p>Cathecolamine contents detected in SH-SY5Y and BE(2)-M17 cell lines before and after differentiation for 7 days.</p

Aggregation studies.

<p>aS (A) and aS/DHA oligomers (B) aggregation process at 37°C in the presence of lipids, followed by ThT binding assay. aS or aS oligomers were dissolved in 20 mM Tris, 150 mM NaCl pH 7.4 at a 50 µM concentration in order to induce aggregation, in the absence (black circles) and in the presence of DOPG SUV, at molar ratio 1:20 (empty circles), 1:50 (empty triangles) and in the presence of DHA (molar ratio 1:50, inverted black triangles). The excitation wavelength was fixed at 440 nm, and the fluorescence emission was collected at 485 nm. To better visualize the aggregation trend of aS and aS in the presence of DOPG (molar ratio 1:20), the data points are fitted with a sigmoidal equation (SigmaPlot software). Inset: TEM images of protein material relative to aS and aS/DHA oligomers samples after 9 days of incubation in the presence of DOPG SUV (molar ratio 1:20).</p

Cellular morphology of undifferentiated and differentiated SH-SY5Y and BE(2)-M17 cells after 7 days of differentiation.

<p>Representative phase contrast images showed that after 7 days of treatment, staurosporine and RA promoted the most remarkable neurite extensions in SH-SY5Y cells and BE(2)-M17 cells, respectively. Scale bar = 100 μM.</p