Journal:

Article Title: Spirochaeta aurantia Has Diacetyl Chloramphenicol Esterase Activity

doi:

Figure Lengend Snippet: MICs of chloramphenicol and diacetyl chloramphenicol for E. coli , two strains of S. aurantia , and three species of Streptomyces

Article Snippet: The bacteria used were S. aurantia M1 (ATCC 25082), S. aurantia J1 ( 5 ), Escherichia coli XL1-Blue MRF′ (Stratagene, La Jolla, Calif.), E. coli pGOΔ1 ( 28 ), Bacillus subtilis EUR9030 ( 6 ), Streptomyces coelicolor A3(2) 2612 ( 13 ), Streptomyces lividans 66 TK24 ( 14 ), and Streptomyces griseus SKK821 ( 1 ).

Techniques:

Journal:

Article Title: Spirochaeta aurantia Has Diacetyl Chloramphenicol Esterase Activity

doi:

Figure Lengend Snippet: Nondenaturing 7.5% polyacrylamide gel electrophoresis and colorimetric analysis for esterase activity in S. aurantia M1 and J1 and in E. coli. Carboxylesterase activity was detected in situ in the gels with α-napthyl acetate and Fast Blue RR (A) or with 4-methylumbelliferyl acetate (B). The bands in the gel shown in panel B were visualized under fluorescent light. Molecular sizes for the markers phosphorylase b (97 kDa), bovine serum albumin (68 kDa), and ovalbumin (43 kDa) are shown to the left of the gels.

Article Snippet: The bacteria used were S. aurantia M1 (ATCC 25082), S. aurantia J1 ( 5 ), Escherichia coli XL1-Blue MRF′ (Stratagene, La Jolla, Calif.), E. coli pGOΔ1 ( 28 ), Bacillus subtilis EUR9030 ( 6 ), Streptomyces coelicolor A3(2) 2612 ( 13 ), Streptomyces lividans 66 TK24 ( 14 ), and Streptomyces griseus SKK821 ( 1 ).

Techniques: Polyacrylamide Gel Electrophoresis, Activity Assay, In Situ

Journal: eLife

Article Title: Genome-wide screen reveals Rab12 GTPase as a critical activator of Parkinson’s disease-linked LRRK2 kinase

doi: 10.7554/eLife.87098

Figure Lengend Snippet: ( A–D ) Microscale thermophoresis of Rab12 binding to fluorescently labeled LRRK2 Armadillo domain (residues 1–552) wild type ( A ) or bearing the indicated mutations at Site #1: K439E ( B ) or Site #3: E240R ( C ) and F283A ( D ). Purified Rab12 was serially diluted and then NHS-RED-labeled-LRRK2 Armadillo (final concentration 100 nM) was added. Graphs show mean and SEM from two independent measurements, each the average of two replicate runs. ( E ) Immunoblot of anti-FLAG antibody immunoprecipitation of FLAG-LRRK2 wild type or indicated Site #3 mutants with endogenous or co-expressed HA-Rab12 protein in HEK293 cells. Lysate inputs (1.5%) are shown at left; membranes were probed with anti-FLAG or anti-Rab12 antibodies. ( F ) Quantitation of two independent experiments carried out in duplicate as in ( E ). ****p<0.0001 for LRRK2 E240R and S244R relative to LRRK2 WT by one-way ANOVA. ( G ) Immunoblot analysis of 293T cells transfected with LRRK2 R1441C or K17/18A R1441G and GFP, GFP-Rab8, or GFP-Rab12 for 36 hr; +/-MLi2 (200 nM for 2 hr). ( H ) Quantitation of the fraction of phosphorylated Rab10 from immunoblots as in ( G ) normalized to respective total Rab10 levels, normalized to LRRK2 R1441C+GFP-Rab12. Error bars indicate SEM from two independent experiments; **p=0.003 for LRRK2 R1441C GFP and GFP-Rab12, **p=0.0044 for LRRK2 K17/18A R1441G GFP and GFP-Rab12, ns = 0.6 by Student’s t -test. Figure 8—source data 1. Raw/annotated gels for .

Article Snippet: Recombinant DNA reagent , pCMV5 Flag-LRRK2 K17/18 A R1441G , Addgene RRID: Addgene_186012 , 186012 , .

Techniques: Microscale Thermophoresis, Binding Assay, Labeling, Purification, Concentration Assay, Western Blot, Immunoprecipitation, Quantitation Assay, Transfection

Journal: eLife

Article Title: Genome-wide screen reveals Rab12 GTPase as a critical activator of Parkinson’s disease-linked LRRK2 kinase

doi: 10.7554/eLife.87098

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pCMV5 Flag-LRRK2 K17/18 A R1441G , Addgene RRID: Addgene_186012 , 186012 , .

Techniques: Bioprocessing, Generated, Labeling, Recombinant, Software