Journal: Human Molecular Genetics
Article Title: PARK2 enhancement is able to compensate mitophagy alterations found in sporadic Alzheimer's disease
Figure Lengend Snippet: Lysosomal alterations in SAD fibroblasts. ( A ) Quantification of the amount of lysosomes per cell. ( B ) Quantification of the lysosomal acidity represented by the ratio between untreated and bafilomycin (100 nM)-treated cells. The following healthy/AD age-matched samples were used: AG1136/AG05809, AG07803/AG06262 and AG05813/AG06263. Every measurement was performed in triplicate, and the graphs represent the means and standard deviations of the average of every cell line. ( C ) Representative western blot of LAMP1 protein in the absence or presence of CCCP (20 μM) and quantification of the levels under basal conditions. The following couples were used: AG11362/AG05809, AG05813/AG06263 and AG07310/AG06869. ( D ) Representative western blot analysis of the protein levels of Cathepsin B/CTSB and quantification of the protein levels of CTSB under basal conditions. The following four couples were used in this assay: AG11362/AG05809, AG07803/AG06262, AG05813/AG06263 and AG07310/AG06869. ( E ). The activity was normalized by the maximum fluorescence detected in the last time point of the healthy sample. The couples used for this experiment were: AG11362/AG05809, AG05813/AG06263 and AG07310/AG06869. Every measurement was performed in triplicate, and the average obtained for each cell line was used for the graph. ( F ) Representative western blot analysis of SQSTM1 and quantification of the levels in the presence of NH 4 Cl with respect to the absence. The following couples were used: AG11362/AG05809, AG07310/AG06869 and AG07803/AG06262. ( G ) Representative confocal microscopy images of healthy and AD fibroblasts expressing mRFP-EGFP-LC3. ( H ) Quantification of autophagolysosomes per cell corresponding to the percentage of only red structures. The couples used for this experiment were: AG11362/AG05809, AG05813/AG06263 and AG07310/AG06869. All graphs show means and standard deviations of the mentioned healthy/AD age-matched couple samples. † P
Article Snippet: The primary antibodies used were: TOMM20 (sc-11415, Santa Cruz Biotechnology, Santa Cruz, C A, USA), COX4I1 (complex IV) (459600, Invitrogen, Eugene, OR, USA), LC3 (B7931, Sigma, St. Louis, MO, USA, for WB), LC3 (M152-3, MBL, Woburn, MA, USA, for immunostaining), SQSTM1 (610832, BD Biosciences, San Jose, CA, USA), BECN1 (sc-11427, Santa Cruz), Ubiquitin (Z0458, Dako, Glostrup, Denmark), LAMP1 (611042, BD Biosciences), CTSB (AB4064, Chemicon, Darmstadt, Germany), GAPDH (ab8245, Abcam, Cambridge, UK), PARK2 (sc-32282, Santa Cruz), PINK1 (BC100-494, Novus, Cambridge, UK) and SCaMC-1 [short calcium-binding mitochondrial carrier 1, kindly given by Dr Araceli del Arco ( )].
Techniques: Western Blot, Activity Assay, Fluorescence, Confocal Microscopy, Expressing