Journal:

Article Title: Cloning of an Intracellular Poly[ d (-)-3-Hydroxybutyrate] Depolymerase Gene from Ralstonia eutropha H16 and Characterization of the Gene Product

doi: 10.1128/JB.183.1.94-100.2001

Figure Lengend Snippet: Detection of PhaZ in R. eutropha H16. SDS-polyacrylamide gel electrophoresis of whole cells and PHB granules from R. eutropha was carried out. The gels were stained with Coomassie brilliant blue R-250 (A) or immunostained (Western blot) (B). Lanes: a-0, whole cells grown for 2 days in nitrogen-rich medium (91 μg of protein); a-1, whole cells after 1 day of PHB synthesis (86 μg of protein, 21 μg of PHB); a-3, whole cells after 3 days of PHB synthesis (88 μg of protein, 73 μg of PHB); b-1, PHB granules after 1 day of PHB synthesis (19 μg of protein, 160 μg of PHB); b-3, PHB granules after 3 days of PHB synthesis (20 μg of protein, 200 μg of PHB); c-1, supernatant fraction of cells after 1 day of PHB synthesis (91 μg of protein); c-3, supernatant fraction of cells after 3 days of PHB synthesis (57 μg of protein); Ni, purified His-tagged depolymerase (0.1 μg of protein); M, molecular mass markers. Sizes are indicated in kilodaltons.

Article Snippet: After metal chelating purification, the protein was finally purified by a continuous elution sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Prep cell model 491; Bio-Rad).

Techniques: Polyacrylamide Gel Electrophoresis, Staining, Western Blot, Purification

Journal:

Article Title: Metabolic Instability of Escherichia coli Cyclopropane Fatty Acid Synthase Is Due to RpoH-Dependent Proteolysis

doi:

Figure Lengend Snippet: Proteolytic degradation of CFA synthase. Autoradiograms show SDS-polyacrylamide gel separations of immunoprecipitates of pulse-chase experiments with various strains. Three independent gels are shown (lanes 1 to 4, 5 to 9, and 10 to 14). Lanes 1 to 3 are extracts of strain YYC1314 (which contains only the chromosomal cfa gene) growing with glycerol as carbon source. Lane 1, extracts from the strain labeled for 40 min without chase; lanes 2 and 3, the same as lane 1, but chases were for 40 and 80 min, respectively. Lane 4 is strain YYC1168 carrying plasmid pAYW19 and was used as a CFA synthase standard (Materials and Methods). Lanes 5 to 9 are extracts of strains grown with glucose as carbon source. Lane 5, cfa null mutant, YYC1317; lanes 6 to 9, extracts of the cfa plasmid-containing strain YYC1318 labeled for 30 min and chased for 0, 30, 60, and 90 min, respectively. Lanes 11 to 14 are extracts from experiments done on strain YYC1318 grown with glucose and labeled for 30 min followed by chase periods of 0, 60, 90, and 120 min, respectively. Lane 10 is a CFA synthase marker in which CFA synthase was labeled in the presence of rifampin in strain YYC1321 (Materials and Methods). The arrows indicate the protein band corresponding to CFA synthase (note that the standard in lane 4 differs from that used in lane 10 and elsewhere in this study). The experiments were done with protocol A (Materials and Methods). Note that residual labeling with cysteine occurred during the chase (see text). Abbreviations: bgrd, background (cfa null mutant); std, standard.

Article Snippet: The purified CFA synthase was further purified by on a preparative SDS-polyacrylamide gel (BioRad model 491 Prep Cell; 14-cm length and 2.8-cm diameter; 10% running and 4% stacking gel).

Techniques: Pulse Chase, Labeling, Plasmid Preparation, Mutagenesis, Marker

Journal:

Article Title: Metabolic Instability of Escherichia coli Cyclopropane Fatty Acid Synthase Is Due to RpoH-Dependent Proteolysis

doi:

Figure Lengend Snippet: Effect of temperature on degradation of CFA synthase. (A) Autoradiograms of SDS-polyacrylamide gel separations of immunoprecipitates of pulse-chase experiments with various strains are shown. The cfa plasmid carrying strain YYC1318 was grown on glucose minimal medium supplemented with methionine and cysteine and labeled as described in Materials and Methods. In each case, the cultures were labeled with radioactive methionine for 30 min at 37°C before initiation of the chase period. In lanes 2 and 3, the chase experiments were done at 30°C. Lane 1, no chase; lane 2, 30-min chase; lane 3, 60-min chase. Lanes 4 and 5 were chased at 37°C for 30 and 60 min, respectively. Lane 6 was chased for 5 min at 42°C and then for 30 min at 37°C. Lane 7 is the CFA synthase marker. Arrows indicate the CFA synthase band. std, standard. (B) Turnover of CFA synthase of strain YYC1318 at 30°C (open circles) and 37°C (filled circles). Each point represented the result from one lane of an experiment such as that of panel A. The experiments were done with protocol B (Materials and Methods).

Article Snippet: The purified CFA synthase was further purified by on a preparative SDS-polyacrylamide gel (BioRad model 491 Prep Cell; 14-cm length and 2.8-cm diameter; 10% running and 4% stacking gel).

Techniques: Pulse Chase, Plasmid Preparation, Labeling, Marker

Journal:

Article Title: Metabolic Instability of Escherichia coli Cyclopropane Fatty Acid Synthase Is Due to RpoH-Dependent Proteolysis

doi:

Figure Lengend Snippet: Effect of mutations in rpoH, lon, clpP, or groEL on degradation of CFA synthase. Autoradiograms of SDS-polyacrylamide gel separations of immunoprecipitates of pulse-chase experiments with various strains are shown. Three gels are shown (lanes 1 to 5, 6 to 12, and 13 to 16). Lanes 2 and 3 are extracts of strain YYC1324 (wild type [wt]) labeled with [35S]methionine for 40 min at 30°C and then chased for 0 (lane 2) or 90 (lane 3) min at 30°C. Lanes 4 and 5 are identical to lanes 2 and 3, respectively, except that the strain labeled was YYC1325 (rpoH). Lanes 6 and 7 are strain YYC1328 (wild type), lanes 8 and 9 are strain YYC1329 (lon), and lanes 10 and 11 are strain YYC1332 (lon clpP). In each case, the strain was labeled for 30 min at 37°C followed by no chase (lanes 6, 8, and 10) or by chase for 60 min at 37°C (lanes 7, 9, and 11). In lanes 13 to 16, the experiments were done at 30°C. Lanes 13 and 14 were strain YYC1336 (wild type), whereas lanes 15 and 16 were from strain YYC1338 (groEL). In each case, the odd-numbered lane received no chase period, whereas the even-numbered lane was chased for 60 min (labeling was done for 30 min). Lanes 1 and 12 contain the CFA synthase marker, and the arrows denote the CFA synthase protein band. The experiments were done using protocol A (Materials and Methods). std, standard.

Article Snippet: The purified CFA synthase was further purified by on a preparative SDS-polyacrylamide gel (BioRad model 491 Prep Cell; 14-cm length and 2.8-cm diameter; 10% running and 4% stacking gel).

Techniques: Pulse Chase, Labeling, Marker

Journal:

Article Title: Metabolic Instability of Escherichia coli Cyclopropane Fatty Acid Synthase Is Due to RpoH-Dependent Proteolysis

doi:

Figure Lengend Snippet: Degradation of E. coli CFA synthase is independent of energy. Autoradiograms of SDS-polyacrylamide gel separations of immunoprecipitates of pulse-chase experiments are shown. Lane 1 is the CFA synthase marker, lanes 2 to 5 were from strain YYC1318, and lane 6 is the cfa null strain YYC1317. Strain YYC1318 was pulse-labeled for 30 min without chase (lane 2) or chased for 30 min in the presence of glucose (lane 3). Lanes 4 and 5 were the same as lane 3 except that the chase was done in the absence of glucose without or with 1 mM KCN, respectively. Arrows denote the CFA synthase protein band. The experiments were done with protocol B (Materials and Methods). Abbreviations are as in Fig. ​Fig.11.

Article Snippet: The purified CFA synthase was further purified by on a preparative SDS-polyacrylamide gel (BioRad model 491 Prep Cell; 14-cm length and 2.8-cm diameter; 10% running and 4% stacking gel).

Techniques: Pulse Chase, Marker, Labeling

Journal: iScience

Article Title: Type 2 diabetes is associated with increased circulating levels of 3-hydroxydecanoate activating GPR84 and neutrophil migration

doi: 10.1016/j.isci.2022.105683

Figure Lengend Snippet: 3-Hydroxydecanoate signals through Gα i , Gα q , and GPR84 (A) Baseline normalized cell index (CI) of cells stimulated with 3-hydroxydecanoate (3-OH-C10) or the positive control compound, zaprinast, in the xCELLigence assay (N = 3). (B–D) Maximum baseline normalized CI of cells stimulated with 3-hydroxydecanoate after pre-incubation with (B) the Gα i inhibitor PTX (200 ng/mL) for 16 h (N = 3–4), (C) the Gα q inhibitor YM-254890 (2 μM) for 15 min (N = 5–7), or (D) the β-arrestin inhibitor barbadin (1 μM) for 15 min (N = 3–4). (E and F) PRESTO-Tango luciferase assay in HTLA cells transfected with human GPR84. (E) Transfected cells were stimulated with 3-hydroxydecanoate, the endogenous GPR84 agonist decanoate (capric acid, C10), the GPR84 reference agonist embelin or the GPR84 antagonist AR505962 (N = 4–5). (F) Transfected cells were pre-incubated with the GPR84 antagonist AR505962, followed by stimulation with sub-maximal concentrations of 3-hydroxydecanoate, decanoate, or embelin (N = 3–4). (G) PRESTO-Tango assay in HTLA cells transfected with human GPR109B (HCA3). The cells were stimulated with 3-hydroxydecanoate, the endogenous GPR109B agonist 2-hydroxyoctanoate (2-OH-C8), or the GPR109B reference agonist AR231783. N = 3. Data are shown as mean ± SEM The logEC50 values were calculated in GraphPad Prism using nonlinear regression.∗p < 0.05, ∗∗p < 0.01. p values were determined by one-way ANOVA followed by Tukey’s multiple comparison test (B–D). See also Figure S3 .

Article Snippet: For antagonist mode, the cells were pre-incubated with 2 μM YM-254890 (FUJIFILM Wako Chemicals #257–00631) or 10 μM barbadin (Toronto Research Chemicals #B118250) for 15 min with readings performed every 15 s; followed by agonist stimulation as described.

Techniques: Positive Control, Incubation, Luciferase, Transfection