Neurons were lysed in 1% SDS lysis buffer, sonicated twice at 3.5 power for 5 seconds and the protein quantified using the bicinchoninic acid assay (BCA). Equal amounts of protein were loaded into freshly cast bis-trisacrylamide gels (10%) and proteins were separated by gel electrophoresis then transferred to PVDF membranes. Membranes were blocked in TBST (5% milk) for 1 hour, then probed overnight at 4°C with primary antibodies for synaptophysin (1:500), or PSD-95 (Neuromab, 1:500). Membranes were incubated for 1 hour with HRP-conjugated secondary antibodies (1:1000) and developed. Band densitometry was determined using Image J software, and results normalized to total protein as determined by Coomassie blue protein stain.
For measurement of astrocyte TSP1 levels, astrocytes were plated on PDL (40 μg/mL) pre-coated 100 mm plates at a density of 2.5 × 106 per plate and cultured for 4 days, serum-deprived for 24 hours, then treated for 24 hours with carbachol (1 mM) prepared in serum free medium. Medium was collected and cells were lysed in 1% SDS lysis buffer from treated and untreated plates immediately after the 24h treatment (time 0), and 6 and 24 hours after treatment washout. Intracellular protein was quantified from lysate samples using the BCA method and equal amounts were loaded into 3–8% Tris-Acetate gels and separated by gel electrophoresis. After transfer, PDVF membranes were blocked in TBST (3% BSA) for 1 hour and probed overnight in goat anti-mouse TSP1 (1:1000), then incubated for 1 hour with HRP-conjugated secondary antibodies (1:1000) and developed. TSP1 was detected by chemiluminescence and normalized to β-actin levels using densitometric analysis. For measurements of TSP1 in the astrocytic medium, 7 mL of media from each time point was concentrated to 200 μl using Pierce concentrators (MWCO 9kDa) centrifuged at 4000 × g for 25 minutes at 25 C. After the addition of protease inhibitors, reducing reagents, and sample buffer, samples were denatured by heating (70°C for 10 minutes) and equal volumes (30 μl) of concentrated sample were loaded into 3–8% Tris-Acetate pre-cast gels. After transfer, PDVF membranes were blocked in TBST (3% BSA) for 1–3 hours and incubated overnight in goat anti-mouse TSP1 (1:500). Band densitometry was determined using Image J software and normalized to corresponding cell lysate protein concentrations.
For measurement of astrocyte TSP1 levels, astrocytes were plated on PDL (40 μg/mL) pre-coated 100 mm plates at a density of 2.5 × 106 per plate and cultured for 4 days, serum-deprived for 24 hours, then treated for 24 hours with carbachol (1 mM) prepared in serum free medium. Medium was collected and cells were lysed in 1% SDS lysis buffer from treated and untreated plates immediately after the 24h treatment (time 0), and 6 and 24 hours after treatment washout. Intracellular protein was quantified from lysate samples using the BCA method and equal amounts were loaded into 3–8% Tris-Acetate gels and separated by gel electrophoresis. After transfer, PDVF membranes were blocked in TBST (3% BSA) for 1 hour and probed overnight in goat anti-mouse TSP1 (1:1000), then incubated for 1 hour with HRP-conjugated secondary antibodies (1:1000) and developed. TSP1 was detected by chemiluminescence and normalized to β-actin levels using densitometric analysis. For measurements of TSP1 in the astrocytic medium, 7 mL of media from each time point was concentrated to 200 μl using Pierce concentrators (MWCO 9kDa) centrifuged at 4000 × g for 25 minutes at 25 C. After the addition of protease inhibitors, reducing reagents, and sample buffer, samples were denatured by heating (70°C for 10 minutes) and equal volumes (30 μl) of concentrated sample were loaded into 3–8% Tris-Acetate pre-cast gels. After transfer, PDVF membranes were blocked in TBST (3% BSA) for 1–3 hours and incubated overnight in goat anti-mouse TSP1 (1:500). Band densitometry was determined using Image J software and normalized to corresponding cell lysate protein concentrations.
Free full text: Click here
