Fig. 4

Extracellular αSYN is internalized by astrocytes in a TLR4 independent manner. Primary astrocytes from littermate Tlr4 ^+/+ and Tlr4 ^−/− mice (a) or Tlr4 ^+/+ mice (b) were continuously treated for 48 h with indicated concentrations of recombinant human synucleins or LPS or left untreated (Ø). Cells were washed twice with PBS prior to lysis. The protein lysates were immunoblotted and probed with human-specific anti-αSYN (a, b) and also antibody against αSYN phosphorylated at S129 (b). GAPDH serves as control for equal protein loading throughout. Positive control in (b) is brain lysate from a 18 month old Thy1[A30P]hSNCA transgenic mouse, which has high amounts of phosphorylated αSYN. N is indicated in the figure (a) or N = 8 (b, c). c Primary wild type astrocytes were treated continuously with 0.7 µM αSYN for indicated times. Protein lysates were examined by immunoblotting as above