Mouse Netrin 1. pMT23 was generously supplied by Dr Thomas Jessell. Dissociated dI neuron culture Embryonic day 13 rat dorsal spinal cord was dissected as previously described in L15 medium and dissociated in 0. 35% trypsin 0. 2% glucose PBS P S at 37 C for 15 minutes. Following trypsin digestion, the tissue was resuspended in culture medium and gently triturated. The single cell suspension was plated in culture medium onto poly D lysine laminin coated tissue culture dishes or 12 mm glass coverslips and incubated overnight at 37 C, 5% CO2. When necessary, dissociated dI neuron cultures were serum starved by incubation in non supplemented F12 medium for 2 h at 37 C.
Immunolabeling of dI neurons Dissociated dI neuron cultures have been fixed in 4% parafor maldehyde PBS for ten minutes, washed as soon as in PBS, blocked and labeled with major and Cy3 conjugated secondary antibodies diluted in 1% heat inactivated goat serum or FBS 0. 1% Triton X one hundred PBS. For nuclear co labeling, DAPI was added with p38-alpha inhibitor the secondary antibody. Coverslips were mounted onto glass microscope slides in Vectashield. Phosphorylation assays For phosphorylation assays, serum starved dissociated dI neuron cultures have been treated with either 4 mM HCl 0. 1% BSA, BMP7 or BMP6, diluted in non supplemented F12 medium in the indicated concentra tions before immunolabeling having a pSmad1 5 8 or a pAkt or preparation of cell lysates for western blot ana lysis. BMP treated explants had been labeled with a phos pho Smad1 five 8. Western blot evaluation Entire cell lysates of dI neuron cultures had been ready using 1x lysis buffer supple mented with 1 mM PMSF.
Samples had been separated by SDS Web page and transferred to nitrocellulose. Nitrocellulose selleck membranes had been blocked in 5% nonfat milk 0. 1% Tween 20 TBS and probed overnight with principal antibodies diluted in 5% BSA 0. 1% Tween 20 TBS, except for the a BMPRII along with a ActRIIB monoclonal antibodies, which were diluted in blocking buffer. Mem branes have been washed in TBST and probed with HRP conjugated secondary antibo dies in blocking buffer. Immediately after washing in TBST, blots had been developed applying the Supersignal West Pico chemoluminescent substrate detection kit and exposed to Kodak BioMax Light Film. For phosphorylation assays, mem branes were washed in TBS, stripped for 30 minutes at 70 C in stripping buffer, washed in TBS and reprobed working with antibodies that recognize total cellular Smad1 5 8 or Akt for normalization from the phosphorylated signals.
The films were imaged making use of the Kodak Digital Science Image Station 440CF and densitometric analysis was performed utilizing ImageJ v1. 37 software program. Development cone collapse assay Serum starved dissociated dI neuron cultures were pre incubated with DMSO, PI3K inhibitors or DM, diluted in non supplemented F12 medium, and then stimulated with 50 ng ml BMP7 or treated with four mM HCl 0. 1% BSA for 30 minutes.