furin mouse monoclonal antibody  (Proteintech)

Journal: Biology Open

Article Title: Intracellular trafficking of furin enhances cellular intoxication by recombinant immunotoxins based on Pseudomonas exotoxin A

doi: 10.1242/bio.061792

Figure Lengend Snippet: HEK293 FRT furin knockout. HEK293 FRT cells were transfected with plasmids containing genes coding for Cas9 and three furin sgRNAs. Surviving cells were clonally selected by serial dilution in a 96-well plate. Lysates from HEK293 FRT (WT) and four mutant clones were evaluated for furin expression by western blot. Clones 1, 2, and 4 showed no discernable furin expression, and clone 1 (boxed) was selected for further study as ΔFur293.

Article Snippet: For furin expression level analysis, mouse anti-furin antibody (mAb, Santa Cruz Biotechnology, sc-133141), mouse anti-β-actin (mAb, Thermo Fisher Scientific, BA3R), and goat anti-mouse IgG alkaline phosphatase conjugated antibody (mAb, Santa Cruz Biotechnology: sc-2058) were used.

Techniques: Knock-Out, Transfection, Serial Dilution, Mutagenesis, Clone Assay, Expressing, Western Blot

Journal: Biology Open

Article Title: Intracellular trafficking of furin enhances cellular intoxication by recombinant immunotoxins based on Pseudomonas exotoxin A

doi: 10.1242/bio.061792

Figure Lengend Snippet: Cleavage assays. HEK293 FRT cells, ΔFur293 cells, and ΔFur293 cells stably expressing transgenic wild-type furin (ΔFur293/Fur) were incubated for various time intervals from 0.5 to 8 h in culture with the anti-transferrin receptor/PE24 RIT HB21-LR. Whole cell lysates were evaluated for full length and cleaved HB21-LR by western blot (panel A) and densitometry (panel B) as described. Also shown are untreated (U) cell lysates for each cell line, HB21-LR with (+) and without (−) furin treatment in vitro , and the β-actin loading control. The ratio between the furin-cleaved band intensity and the total intensity of all RIT bands at each time point is plotted in panel B. The individual densitometric analysis values (points) and mean (bar) for at least two separate assays of each cell line are shown.

Article Snippet: For furin expression level analysis, mouse anti-furin antibody (mAb, Santa Cruz Biotechnology, sc-133141), mouse anti-β-actin (mAb, Thermo Fisher Scientific, BA3R), and goat anti-mouse IgG alkaline phosphatase conjugated antibody (mAb, Santa Cruz Biotechnology: sc-2058) were used.

Techniques: Stable Transfection, Expressing, Transgenic Assay, Incubation, Western Blot, In Vitro, Control

Journal: Biology Open

Article Title: Intracellular trafficking of furin enhances cellular intoxication by recombinant immunotoxins based on Pseudomonas exotoxin A

doi: 10.1242/bio.061792

Figure Lengend Snippet: Complementation with mutant furin. ΔFur293 cells were stably transfected with genes for furin that contained mutations designed to impair its intracellular trafficking or catalytic function. Mutations S773A/S775A (ADA) and S773D/S775D (DDD) alter furin trafficking, while the N295A mutant (Ala-295) inhibits catalytic activity. Two separate clonal lineages stably transfected with mutant or wild-type furin were treated with the anti-transferrin receptor/PE RIT HB21-LR to assess cytotoxicity. The EC50 (pM) values from at least four separate assays for each line were normalized for furin expression levels and plotted. Dashed lines denote the average value for each clone and error bars indicate the standard error. The largest significant P -values ( P <0.05) between sets of clones from a one-way ANOVA performed as described are indicated. All P -values are reported in Table S3 .

Article Snippet: For furin expression level analysis, mouse anti-furin antibody (mAb, Santa Cruz Biotechnology, sc-133141), mouse anti-β-actin (mAb, Thermo Fisher Scientific, BA3R), and goat anti-mouse IgG alkaline phosphatase conjugated antibody (mAb, Santa Cruz Biotechnology: sc-2058) were used.

Techniques: Mutagenesis, Stable Transfection, Transfection, Activity Assay, Expressing, Clone Assay

Journal: Biology Open

Article Title: Intracellular trafficking of furin enhances cellular intoxication by recombinant immunotoxins based on Pseudomonas exotoxin A

doi: 10.1242/bio.061792

Figure Lengend Snippet: Cleavage by mutant furin . ΔFur293 cells stably expressing transgenic furin mutants (FurADA, FurDDD, and FurAla-295) were incubated for various time intervals from 0.5 to 8 h in culture with the anti-transferrin receptor/PE24 RIT HB21-LR. Whole cell lysates were evaluated for full length and cleaved HB21-LR by western blot (panel A) and densitometry as described. Also shown are untreated (U) cell lysates for each cell line, HB21-LR in vitro with (+) and without (−) furin treatment, and the β-actin loading control. The ratio between the furin-cleaved band intensity and the total intensity of all RIT bands at each time point is plotted in panel B. The individual densitometric analysis values (points) and mean (bar) for at least two internalization and cleavage assays in each cell line are shown.

Article Snippet: For furin expression level analysis, mouse anti-furin antibody (mAb, Santa Cruz Biotechnology, sc-133141), mouse anti-β-actin (mAb, Thermo Fisher Scientific, BA3R), and goat anti-mouse IgG alkaline phosphatase conjugated antibody (mAb, Santa Cruz Biotechnology: sc-2058) were used.

Techniques: Mutagenesis, Stable Transfection, Expressing, Transgenic Assay, Incubation, Western Blot, In Vitro, Control