Nis element ar analysis 4.20.02 64 bit software

Cells were grown in 12-well–plates, on cover slides that were pre-coated with poly-d-lysine (100 μg/ml, for 1 h). Cells were transfected to express PH-PLCδ1-GFP using Jet-PRIME transfection reagent (Polyplus, France). When indicated, cells were co-transfected with WT α-Syn or one of the specified α-Syn mutations (A30P, E46K, A53T, K10,12E, or K21,23E), or a mock plasmid. In some experiments, cells were conditioned in the presence of 50 μg/ml of 647 concanavalin A (ConA, molecular probes, Invitrogen, Rehovot, Israel) in DMEM, at 37 °C for 10 min to label the plasma membrane. Membranes were defined by the ring-shaped ConA signal around the cell and were differentiated from the cytoplasm. Membrane to cytosolic PH-PLCδ1-GFP signal ratio was calculated using the NIS-Element AR Analysis 4.20.02 64-bit software (Nikon, Agentek, Tel Aviv, Israel) (5 (link)).

HeLa cells were grown on cover slides coated with poly-D-Lysine, in 12-well plates. Cells were co-transfected with PH-PLCδ1-GFP and either WT, A53T or K10,12E α-Syn expressing plasmids, using JetPRIME transfection reagent polyplus (Tamar, Rehovot, Israel). Forty eight hours post transfection, cells were incubated with 50 μg/ml Alexa-647 Concanavalin (Con) A (molecular probes, Invitrogen, Rehovot, Israel) in DMEM, at 37 °C for 10 min; washed in cold serum-free DMEM; and fixed with 4% paraformaldehyde for 10 min, on ice. Cells were then washed one more time and permeabilized with 0.2% Triton X-100 in blocking solution (1.5% BSA in PBS) for 5 min at room temperature. Cells were incubated with anti α-Syn antibody, C20 (Santa Cruz, Dallas TX, US) at 1:500 dilution, overnight at 4 °C, followed by a secondary ab. Membrane to cytosolic signal ratio of PH-PLCδ1-GFP was calculated using the NIS-Element AR Analysis 4.20.02 64-bit software (Nikon, Agentek, Tel Aviv, Israel). Membranes were defined by the ring-shaped ConA signal around the cell and differentiated from the cytoplasm of the cells.