Cytosolic PLA₂ activation in Purkinje neurons and its role in AMPA-receptor trafficking

JCS032987 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. Sustained phosphorylation of cPLA₂α after PMA treatment. (A) Immunocytochemistry with antibody to cPLA₂α phosphorylated at Ser505 and to calbindin D. Purkinje cells were pre-treated with 200 nM PMA for 20 minutes, and then stimulated for 3 minutes with vehicle or 30 µM AMPA. After washing, the cells were incubated for 2 hours at 37°C before fixation. Scale bar: 10 µm. (B) The fluorescence intensity of phosphorylated cPLA₂α in proximal dendrites was measured. The values are means ± s.e.m. (PMA, n=10; PMA and AMPA, n=8).

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. Effect of PMA on AMPA-induced GFP-cPLA₂α translocation. (A) Time-lapse images of GFP-cPLA₂α fluorescence following the local application of a 30 µM AMPA solution (t=1−3 seconds, indicated by the horizontal bar) in the presence of 200 nM PMA, which was bath-applied 10 minutes prior to the addition of AMPA. ΔF/F₀ of GFP-cPLA₂α is displayed according to a pseudocolor scale. Scale bar: 20 µm. (B) Time course of ΔF/F₀ in the dendritic (solid lines) and somatic (dashed lines) Golgi structures measured in the indicated areas in A.

• Movie 1 -

  Movie 1. Time-lapse images of GFP-cPLA₂α fluorescence following the local application of a 30 µM glutamate solution (t=1−5 seconds, marked by Glu). Relative fluorescence intensity (ΔF/F₀) is displayed according to a pseudo-color scale. Images were acquired every 2 seconds.

• Movie 2 -

  Movie 2. Simultaneous imaging of RFP-cPLA₂α translocation and the Ca²⁺ response. Time-lapse images of RFP-cPLA₂α (left panel) and Oregon green 488 BAPTA-1 (OGB-1, right panels) after the application of 30 µM AMPA (for 5 seconds, marked by AMPA) and 300 µM ACPD (for 5 seconds, marked by ACPD). Images were acquired every 5 seconds.