Fig 6. Strain A3 showed higher levels of autophagy upon α-syn expression.

Strains A3, A4 (A), or corresponding strain atg5Δ (B) were grown in liquid SD media lacking a nitrogen source, and cellular lysates were probed with antibody against either GFP, or Pgk1 (loading control). The A3 strain showed increased processing of GFP-Atg8. Only unprocessed GFP-Atg8 was detected in the atg5Δ strain, as expected, because of the inhibition of autophagy. Immunoblotting using anti-Pgk1 antibody was used as a loading control. (C) Indicated strains transformed with empty plasmid, or plasmid encoding inducible α-syn, were grown in inducible media for 12 h. For quantitation, qRT-PCR was performed using primers specific for ATG8, or PGK1 (internal control). As seen, α-syn mediated induction of Atg8 mRNA was higher in strain A3. (D) The plasmid encoding the mCherry-ATG8 fusion protein was co-transformed with p412P_GAL-SYN-GFP (CEN) or p422P_GAL-SYN-GFP (2μ) in strains A2 and A3. A pre-grown culture from SD media was diluted into SGal to a final concentration (O.D._600nm) of 1.0, and further cultured for 12 h at 30°C. Cells were collected, and lysates probed with the indicated primary antibodies. Strain A3 showed reduced α-syn steady-state levels, and increased levels of free mCherry. The immunoblot with anti-GFP antibodies confirms relatively higher degradation of α-syn-GFP into various smaller fragments in A3 strain. Error bars represent the standard error of replicates performed 3 times. P-value were calculated using paired t-test.