Figure 6: Disruption of ROCK1 leads to impaired autophagy invivo.

(a) Genomic structure of ROCK1 (exon 4–6 only) with targeted allele, Flp-deleted allele and Cre-deleted allele. (b) PCR assay using tail genomic DNA from three different genotypes, separated on 0.7% agarose gel (top). Western blot analysis of ROCK1 expression in the heart tissue from three different genotypes using anti-ROCK1 antibody (bottom). β-Actin was used for protein loading control in each lane. (c) Mice were fed or starved for 16 h, and heart samples were prepared in lysis buffer. Resulting proteins were resolved by SDS–PAGE and blotted against ROCK1, LC3, autophagy marker p62 and β-actin. (d) ROCK1-deficient hearts or control hearts from animals fed or starved for 16 h were subjected to electron microscopy to identify autophagosome structures. Arrows indicate autophagic vacuoles. Scale bar, 500 nm. (e) Hearts from fed and starved ROCK1 WT and KO mice were sectioned, fixed in acetone and immunofluorescence assay was performed against endogenous LC3. Data are representative of two independent experiments from each genotype. Scale bar, 50 μm.