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. Author manuscript; available in PMC: 2009 Feb 3.
Published in final edited form as: Nat Chem Biol. 2008 May;4(5):295–305. doi: 10.1038/nchembio.79

Figure 2. Imidazoline-1 receptor agonists act through cAMP to Rap2B and PLC-ε to modulate autophagy.

Figure 2

a, A53T α-synuclein clearance in stable PC12 cells as in Fig. 1a, treated with or without 1 μM rilmenidine, 1 mM dibutyl-cAMP (db-cAMP), 24 μM forskolin or 500 μM 2′5′dideoxyadenosine (2′5′ddA) for 24 h. Note that control clearance levels for a given substrate may vary from figure to figure, due to different exposures of blots, which aid illustration of agents that either increase or retard clearance.

b, The proportions of EGFP-positive cells with EGFP-HDQ74 aggregates in SK-N-SH (for rilmenidine) and COS-7 (for db-cAMP, forskolin, 2′5′ddA) as in Fig. 1c, treated with concentrations used in Fig. 2a for 48 h post-transfection. Error bars: 95% confidence interval.

c, Endogenous LC3-II levels in PC12 cells treated with 1 μM rilmenidine or 500 μM 2′5′ddA for 24 h.

d, A53T α-synuclein clearance in stable PC12 cells as in Fig. 1a, treated with or without 8-CPT-2Me-cAMP or 6-Bnz-cAMP (1 μM or 10 μM).

e, The proportions of EGFP-positive COS-7 cells with aggregates as in Fig. 1c, treated with or without 10 μM 8-CPT-2Me-cAMP or 6-Bnz-cAMP for 48 h post-transfection. Error bars: 95% confidence interval.

f, COS-7 cells, transfected for 4 h with EGFP-HDQ74 along with empty vector (pcDNA3.1), dominant-negative S17N Rap2B (DN Rap2B) or wild-type PLC-ε (1:3 ratio), were assessed for the proportions of EGFP-positive cells with aggregates after 48 h. Error bars: 95% confidence interval.

g, Endogenous LC3-II levels in COS-7 cells transfected with a dominant-negative Rap2B (DN Rap2B) for 4 h were assessed after 48 h expression.

h, The proportions of EGFP-positive COS-7 cells with aggregates, transfected with EGFP-HDQ74 along with empty vector (pcDNA3.1) or dominant-negative Rap2B (1:3 ratio) for 4 h, were treated with or without 10 μM 8-CPT-2Me-cAMP for 48 h. Error bars: 95% confidence interval.

i, COS-7 cells, transfected with EGFP-HDQ74 and dsRed2 or cytosolic dsRed2-IP3 kinase A (1:3 ratio) for 4 h, were assessed at 48 h post-transfection for aggregation and cell death (as in Fig. 1c). Error bars: 95% confidence interval. ***, p<0.001; **, p<0.01; *, p<0.05; NS, Non-significant.