Proximity Ligation Assays (PLAs) to detect IP3R1-VDAC1 or VAPB-PTPIP51 interactions were performed as described previously using Duolink In Situ Orange Kits (Sigma) according to manufacturer’s instructions [17 (link), 18 (link), 35 (link)]. After amplification steps, samples were immunostained with β-tubulin III (SHSY5Y cells) or MAP2 (neurons) to confirm neural identity.
To detect serine-9 phosphorylated GSK3β, cells were fixed in 4% paraformaldehyde, washed three times in Tris-HCl buffered saline (TBS), permeabilised with 0.1% Triton X-100 in TBS for 15 min and blocked in 5% bovine serum albumin (BSA) in TBS for 1 h. Cells were then labelled with primary antibody diluted in 1% BSA in TBS overnight at 4 °C, washed three times with 0.01% Triton X-100/TBS, incubated with secondary antibodies diluted in TBS, washed three times with TBS and mounted in aqueous mounting medium with DAPI (Abcam). Z-plane images with 0.3 μm intervals were captured using a Nikon Eclipse Ti-E Inverted microscope with CFI Apo Lambda S 60x/1.40 objective and an Andor iXon EMCCD camera equipped with Visitech iSIM Super Resolution System. Mean cytoplasmic fluorescent intensities were quantified using ImageJ.
To detect serine-9 phosphorylated GSK3β, cells were fixed in 4% paraformaldehyde, washed three times in Tris-HCl buffered saline (TBS), permeabilised with 0.1% Triton X-100 in TBS for 15 min and blocked in 5% bovine serum albumin (BSA) in TBS for 1 h. Cells were then labelled with primary antibody diluted in 1% BSA in TBS overnight at 4 °C, washed three times with 0.01% Triton X-100/TBS, incubated with secondary antibodies diluted in TBS, washed three times with TBS and mounted in aqueous mounting medium with DAPI (Abcam). Z-plane images with 0.3 μm intervals were captured using a Nikon Eclipse Ti-E Inverted microscope with CFI Apo Lambda S 60x/1.40 objective and an Andor iXon EMCCD camera equipped with Visitech iSIM Super Resolution System. Mean cytoplasmic fluorescent intensities were quantified using ImageJ.
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