Regulation of PI3K signalling by the phosphatidylinositol transfer protein PITP during axonal extension in hippocampal neurons

JCS019166 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. Representative blots demonstrating levels of PITPα and PITPβ expression in different rat tissues. For quantification of PITPα and PITPβ expression (see Fig. 1), different rat tissues were lysed in RIPA buffer in the presence of cell protease inhibitors and centrifuged at 15000 g for 30 minutes at 4°C to remove cell debris. Equal concentrations of lysates were then separated by SDS-PAGE and proteins were transferred onto nitrocellulose. Level of expression was quantified by comparing band density of PITPα and PITPβ with defined concentration of bacterially expressed recombinant proteins, using His-tagged PITPα and PITPβ-sp1 as standards.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. Nucleofection of hippocampal neurons with PITPα-GFP resulted in a substantial increase in PITPα protein levels in stage 3 neurons. Hippocampal neurons were nucleofected with GFP or PITPα-GFP, and following culture for 36 hours neuronal cell lysates were analysed by immunoblotting using an anti-PITPα antibody.

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. Localization of PITPα-GFP in stage 3 hippocampal neurons. E18 hippocampal neurons were nucleofected with GFP or PITPα-GFP and cultured for 2 days before fixation and analysis. (A) Mean relative fluorescence intensity or GFP or PITPα-GFP in neurites and axonal processes. Each data point is the mean ± s.e.m., n=7. (B) Percentage of hippocampal neurons that showed GFP or PITPα-GFP fluorescence in all processes (ubiquitous) or in the axon only. Each data point is the mean ± s.e.m., n=96, *P<0.0001.

• Supplemental Figure S4 (Adobe PDF) -

  Fig. S4. Overexpression of PITPα does not affect neuronal polarization. E18 hippocampal neurons were nucleofected with indicated constructs and cultured for 1.5 days (A,B) or 4 days (C) before fixation and analysis. (A) PITPα-GFP is highly enriched in the axonal, Tau-1 positive neurite, whereas minor processes exhibit only little PITPα-GFP signals. All neurites are labelled with βIII-tubulin. (B) GFP-positive neurons within different treatment was scored as polarized by the presence of a single, long neurite exhibiting no less than twice the length of the remaining shorter neurites. (C) At day 4, GFP- and PITPα-GFP-expressing neurons were labelled with anti-MAP antibody to visualize dendritic processes. Scale bars: 20 µm in A; 30 µm in B.

• Supplemental Figure S5 (Adobe PDF) -

  Fig. S5. BDNF effects on Akt phosphorylation and axonal branching. (A) E18 hippocampal and cortical neuronal cultures were established as before with the difference that B27 was removed at 1 DIV where indicated. BDNF (50 ng/ml) was applied for indicated times and cell lysates were analysed by western blotting using indicated antibodies. Prolonged stimulation with BDNF activates Akt only in the presence of B27. (B) Hippocampal neurons were nucleofected as previously described and cultured in the presence of BDNF (50 ng/ml). BDNF induces an increase in axonal branching, which was not altered by PITPα siRNA treatment. Data points show the mean ± s.e.m. of three independent experiments (n>60).