Figure 6. Calpain cleaves G_sα creating a link between the cAMP and Ca²⁺-calpain pathways.

a, A53T α-synuclein clearance in stable PC12 cells as in Fig. 1a, treated with or without 100 nM PACAP with or without 10 μM calpastatin or 500 μM 2′5′ddA for 24 h.

b, The proportion of EGFP-positive cells with aggregates in SK-N-SH cells as in Fig. 1c, treated for 48 h with 1 μM PACAP or 200 μM NF449. Error bars: 95% confidence interval.

c, Aggregation in COS-7 cells transfected with either pcDNA3:1 (empty vector) or constitutively active (CA) m-calpain and EGFP-HDQ74 (3:1 ratio) for 4 h and then treated with or without 500 μM 2′5′ddA for 48 h. Error bars: 95% confidence interval.

d, HeLa cells transfected with control or G_sα siRNA for 72 h were analysed for G_sα levels by immunoblotting with anti-G_sα antibody. Aggregation was assessed in HeLa cells transfected with control or G_sα siRNA and EGFP-HDQ74 for 72 h. Error bars: 95% confidence interval.

e, Endogenous LC3-II levels in HeLa cells transfected with control or G_sα siRNA for 72 h.

f, Endogenous LC3-II levels in PC12 cells treated with or without 200 μM NF449 for 24 h.

g, Endogenous LC3-II levels in HeLa cells transfected with control or G_sα siRNA for 72 h and treated with or without 400 nM bafilomycin A1 for the last 4 h. Densitometric analysis is relative to actin. Error bars: Standard error of mean.

h, The proportions of EGFP-positive HeLa cells with aggregates, transfected with control or G_sα siRNA for 48 h, and then re-transfected together with pcDNA 3.1 (empty vector) or constitutively active (CA) m-calpain and EGFP-HDQ74 (3:1 ratio) for a further 48 h. Error bars: 95% confidence interval. ***, p<0.001; **, p<0.01; *, p<0.05; NS, Non-significant.