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. 2022 Oct 20;8:136. doi: 10.1038/s41531-022-00388-7

Fig. 6. Assessment of pS129 antibodies detection in the nuclear, cytosolic, and membranous fractions of aSyn KO and WT primary hippocampal neurons by WB.

Fig. 6

a, b Sequential biochemical extraction was performed on aSyn KO and WT primary hippocampal neurons cultured for 14 days (DIV14), and the pS129 level was assessed in the nuclear (N), cytosolic (C), and membrane (M) fractions (a) or in the soluble (sol) and insoluble (insol) fractions (b) by WB using the six pS129 antibodies (pSyn#64, MJF-R13, 81A, EP1536Y, GTX, and LASH-EGT). Forty nanograms of recombinant aSyn WT or phosphorylated at residue S129 (pS129, indicated by the red arrow) were used as positive controls. The pS129 antibodies were used at a dilution of 1/1000. Membranes were then counterstained by total aSyn antibodies (SYN-1 or Fl-140 α/β/γ synuclein). aTubulin, Laminin B, and E-Cadherin were used as loading control for the cytosolic, nuclear, and membrane fractions, respectively (a). Actin was used as a loading control for the soluble and insoluble fractions (b). All blots were derived from the same experiment and were processed in parallel.