Goat anti rabbit alkaline phosphatase

Cortical neuron cultures from WT and Doc2a, b DKO mice were plated to 200 K cells per well, infected at DIV 1 with lentiviral Doc2b constructs (LIP #1984, #1985, #1986 as above) and harvested at DIV 17. Cells were washed 2 times with PBS, lysed in Laemmli sample buffer, loaded with 50% or the totality of each well for WT and DKO cells respectively, separated by SDS-PAGE and blotted on PVDF membrane (Biorad). Membranes were blocked in 2% skim milk powder (Merck) and 0.5% FCS (Gibco) in PBS with 0.01% Tween-20 (Sigma-Aldrich). Doc2B polyclonal antibody 13.2 was used as primary antibody (1:500) for incubation overnight at 4 °C. Goat anti rabbit alkaline phosphatase (Jackson lab) was used as secondary and Attophos (Promega) as substrate for 30 min incubation at RT. Reprobing was made for actin immunostaining with monoclonal anti-actin antibody C4 (1:3000; Chemicon) and Goat anti mouse alkaline phosphatase as secondary Ab (1:10000).