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. 2016 Nov 2;10:253. doi: 10.3389/fncel.2016.00253

Figure 4.

Figure 4

ROCK1 increases autophagy via an interaction with Beclin1. (A) Increased ROCK1 CA levels induced an increase in the intracellular autophagosome formation and accumulation. SH-SY5Y cells (upper panels) and primary neurons (lower panels) were co-transfected with ROCK1 CA plasmid and mCherry-GFP-LC3B adenovirus (pcDNA was used as a control). Fluorescence microscopy was used to detect the formation of GFP-LC3 puncta after 24 h post-transfection. (B) Expression of Beclin1 following ROCK1 knockdown and ROCK1 CA over-expression. (C) Quantification of Beclin1 following ROCK1 knockdown and ROCK1 CA over-expression. (D) HEK293 cells transfected with ROCK1 CA plasmid and pcDNA. Forty eight hours post-transfection co-immunoprecipitation (CoIP) was performed with cell lysates by using ROCK1 crosslinked agarose and then blotted with antibodies. (E) Representative immunoblot of LC3 II/LC3 I after ROCK1 CA over-expression and ROCK1 knockdown. (F) Quantification of CoIP Beclin1 levels. (G) Representative immunoblot of LC3 II/LC3 I after ROCK1 CA over-expression and ROCK1 knockdown. (H) Representative immunoblot of LC3 II/LC3 I after ROCK1 CA over-expression and ROCK1 knockdown. (I) ROCK1 CA plasmid transfection reverses 3-methyladenine (3-MA) induced autophagy inhibition. Representative immunoblot of LC3 II/LC3 I. (J) Increased Aβ40 secretion after 3-MA treatment (48 h) by ELISA. Data are presented as Mean ± SEM, students’ t test, *p < 0.05, **p < 0.01, ***p < 0.001.