Journal: Molecular cell

Article Title: In vivo X-ray footprinting of pre-30S ribosomes reveals chaperone-dependent remodeling of late assembly intermediates

doi: 10.1016/j.molcel.2013.09.020

Figure Lengend Snippet: (A) Relative quantities of r-proteins in ΔRimM and ΔRbfA pre-30S complexes. For each protein, the ratio of unlabeled (L) peptide in the test sample to 15N-labeled (H) peptide from MRE600 TP30 was determined by data-dependent LC-MS/MS (Experimental Procedures) and normalized to that of protein S8. Error bars represent the standard deviation of three technical replicates. Lower panels compare the relative pre-30S proteins (L/H) to L/H values from wild type (K12) 30S ribosomes. L/H values in the K12 samples ranged from 60–130% and reflected the intrinsic variation in recovery of tryptic peptides (data not shown). R-proteins are organized by their position in the Nomura map and colored by location: white, 5′ domain (body); light gray, central domain (platform); dark gray, 3′ domain (head); black, S1. Protein S1 was readily detected in K12 ribosomes but depleted in pre-30S complexes, consistent with their reduced activity.

Article Snippet: For mass spectrometry, pre-30S proteins were compared to TP30 from E. coli K12 (ATCC).

Techniques: Labeling, Liquid Chromatography with Mass Spectroscopy, Standard Deviation, Activity Assay

Journal: Cell Metabolism

Article Title: Mitochondria and Quality Control Defects in a Mouse Model of Gaucher Disease—Links to Parkinson’s Disease

doi: 10.1016/j.cmet.2013.04.014

Figure Lengend Snippet: Loss of gba Impairs Both Autophagic and Proteasomal Degradation Machinery (A) Autophagic markers from mixed midbrain gba +/+ , gba +/− , and gba −/− cultures were analyzed via western blotting using antibodies as indicated. (B) Autophagic flux was analyzed in gba +/+ and gba −/− midbrain neurons. Neurons were treated with 100 nM bafilomycin A1 and 1 μM rapamycin and flux assayed by comparing LC3I and LC3II levels via western blotting. β-actin was used as a loading control. (C) Densitometry analyzes of autophagy of (B) expressed as a ratio of LC3II/LC3I. Error bars, ± SEM. * p < 0.05. (D) Accumulation of p62/SQSTM1 was analyzed via immunoblotting. β-actin was used as a loading control. (E) Midbrain neurons were treated with either 10 μM MG132 or vehicle control DMSO. Proteins were extracted and analyzed via western blotting and stained with anti-ubiquitin antibodies. (F) Ubiquitin linkage of isolated midbrain analyzed using western blotting with antibodies as indicated. β-actin was used as a loading control. (G) Proteasomal activity (chymotrypsin activity) was measured using the Proteasome Glo assay where activity is proportional to the released luciferase. RFU, relative luciferase units. Data represent the mean ± SEM (n = 3, each experiment contained triplicates for each genotype). * p < 0.05.

Article Snippet: To monitor autophagic flux, cultured midbrain neurons were either treated with 100 nM bafilomycin A1 (Sigma), 1 μM rapamycin (Stress Marq Biosciences), or both drugs in combination for 8 hr at 37°C.

Techniques: Western Blot, Staining, Isolation, Activity Assay, Glo Assay, Luciferase