Journal: PLoS ONE

Article Title: Identification and Characterization of Sulfated Carbohydrate-Binding Protein from Lactobacillus reuteri

doi: 10.1371/journal.pone.0083703

Figure Lengend Snippet: Supernatant, cell surface, and whole cell lysate fractions at different culture times were analyzed by western blotting. Outer surface proteins of L. reuteri JCM1081 were extracted with GHCl. Reactivity with anti-EF-Tu and anti-RNA polymerase antibodies is shown. An anti-RNA polymerase antibody was used to confirm whether cell lysis occurred. Culture time and pH are indicated in the table.

Article Snippet: Anti-RNA polymerase antibodies (1∶2500 dilution; Acris Antibodies, San Diego, CA, USA) were used to verify that cell lysis had occurred.

Techniques: Western Blot, Lysis

Journal: Cell Death & Disease

Article Title: Integrated stress response is critical for gemcitabine resistance in pancreatic ductal adenocarcinoma

doi: 10.1038/cddis.2015.264

Figure Lengend Snippet: Gemcitabine causes translation repression in pancreatic cancer cells. ( a ) PANC-1 cells were incubated for 12 h in the absence or presence of 10 μ M Gem or 0.5 μ M ISRIB. Cell lysates were subjected to sucrose gradient centrifugation, and gradients were fractionated in-line with 254 nm UV absorbance measurement. Top fractions containing free ribosomal subunits are labeled as 40/43S, 60S. Mono ribosomes are labeled as 80S. Ribosomes bound to RNA fractions were labeled as polysomes. ( b ) PANC-1 cells were transfected with an empty vector or the GADD34 plasmid DNA, and incubated 24 h later with 10 μ M Gem for 12 h. Cell lysates were analyzed for polysome profiles as in ( a ). ( c ) PANC-1 cells were incubated in the absence or presence of Gem or ISRIB+Gem and subjected to sucrose gradient centrifugation as in ( a ). Fractions were collected and luciferase (10 ng/ml) spiked into each fraction. RNA Isolated and ATF4 transcript levels were quantitated using qRT-PCR, and normalized to spike-in luciferase control. The percent total ATF4 transcript for each fraction is represented. Fractions 5, 6, and 7 corresponds to fractions with polysomes rich in translation. ( d ) PANC-1 cells were transfected with the TK-ATF4-Luc plasmid construct that with illustrated features: ATF4 5′UTR harboring uORF1 and uORF2, TK promoter and Luciferase coding region. At 24 h post transfection cells were incubated for 12 h in the absence or presence of 10 μ M Gem and 0.5 μ M ISRIB. Firefly luciferase units were measured and normalized to internal control Renilla luciferase activity. Data are the means ±S.D. of three experiments. * P <0.05, ** P <0.005 compared with control, ## P <0.001 compared with Gem+ISRIB. Panels a and b show representative data from three independent experiments

Article Snippet: Synthetic poly (A)+ luciferase RNA (Promega) was spiked (10 ng/ml) into each collected fractions, and RNA was isolated from these fractions using Trizol-LS reagent (Invitrogen).

Techniques: Incubation, Gradient Centrifugation, Labeling, Transfection, Plasmid Preparation, Luciferase, Isolation, Quantitative RT-PCR, Control, Construct, Activity Assay