Supplemental Data
Files in this Data Supplement:
- supplemental figures (.pdf, 774 KB) - Supplemental Fig. 1. PLC��3 knockdown was specifically carried out in cerebellar granule cells. Cultured granule cells transfected with control pGSU6-GFP were immunostained with anti-GFAP antibody, an astrocyte marker. No astrocyte was detected in the primary granule cell culture in the presence of AraC. Bar scale represents 20 ��m. Supplemental Fig. 2. Absence of astrocytes or oligodendrocytes in cortical neuronal culture. Cortical neurons were prepared from E14 mice and cultured for 3 days in the presence of AraC. Neither astrocyte nor oligodendrocyte was detected in the cortical culture as shown by immunostaining with anti-GFAP or anti-O4 antibody. Astrocytes were prepared from P2 mice according to the protocol (20) and primary cell was immunostained by anti-GFAP antibody (red). The validity of anti-O4 antibody (green) was also confirmed by immunohistochemistry of brain section from P2 mice. Anti MAP2 antibody (red) and Hoechst 33342 (blue) were used for cell staining. Supplemental Fig. 3. Effects of phorbol 12-myristate 13-acetate (PMA), thapsigargin, or ionomycin on RhoA expression in Neuro2a. Neuro2a cells were transfected with pENTRTM/U6 vector encoding control or PLC��3 targeting sequence (KD1) and incubated in DMEM without serum in the absence or presence of 100 ng/ml PMA for 48 h, 0.25 ��M thapsigargin (TG) for 24 h, or 0.5 ��M ionomycin (IM) for 24 h. The total amounts of RhoA, PLC��3, and ��-actin were analyzed by immunoblotting. The quantification of RhoA levels was carried out from 4 experiments and the relative values were represented as bar graphs. P���0.05 (Tukey��s mutiple comparison test).