rabbit anti mouse albumin antibody  (Bethyl)

Journal: EMBO Reports

Article Title: An hepatitis B and D virus infection model using human pluripotent stem cell-derived hepatocytes

doi: 10.1038/s44319-024-00236-0

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rabbit anti-FoxA2 (1:400) was purchased from Cell Signaling, mouse anti-AFP (1:1000) from Sigma-Aldrich, mouse anti-ALB (1:1000) from Cedarlane, mouse anti-CD63 from Santa Cruz Biotechnology (1:400), rabbit anti-ADAR1 from Cell Signaling (1:1000), rabbit anti-LDLR from Abcam (1:200), rabbit anti-LAMP1 (1:200) and rabbit anti-EGFR (1:100) from Cell Signaling and rabbit anti-SCARB1 from Novus Biologicals (1:500).

Techniques: Recombinant, Immunofluorescence, Western Blot, Sequencing, Membrane, Knock-Out, Transfection, Reverse Transcription, SYBR Green Assay, Protease Inhibitor, Diagnostic Assay, Software, Microscopy, Imaging

Journal: bioRxiv

Article Title: SUMO chains depolymerization induces slender to stumpy differentiation in T. brucei bloodstream parasites

doi: 10.1101/2023.11.15.567218

Figure Lengend Snippet: (A) Quantification of RNA transcript levels corresponding to the VSG221 gene expressed in wild type (WT) and SUMO all KR parasites. Transcript levels were determined using qRT-PCR and normalized against 7SL and C1. (B) VSG221 expression levels were evaluated by Western blot analysis in total cell extracts with anti-VSG221 antibodies and tubulin as a loading control. Experiments were performed at least in triplicates and one representative image is shown. Quantification of band intensities was performed using ImageJ. (C) The density of the VSG coat in WT and SUMO all KR parasites was analyzed in fixed cells with anti-VSG221 antibodies and flow cytometry. Approximately 50000 events were captured. Representative flow cytometry histograms normalized to mode are shown. MFI: median fluorescence intensity.

Article Snippet: BF cells were collected by centrifugation (1000 x g for 10 min), washed with TDB supplemented with glucose 20 mM and fixed with 4% paraformaldehyde (PFA) in PBS for 1 h. Parasites were allowed to bind to poly-L-lysine coated glass coverslips for 30 min and then incubated with 25 mM NH4Cl for 15 min. Permeabilization and blocking were performed with 3% bovine serum albumin (BSA), 0.5% saponin and 5% normal goat serum in PBS for 1 h. Mouse anti- Tb SUMO (1:500) [ ], mouse anti-VSG221 (1:200), rabbit anti-VSG221 (1:500), mouse anti-EP FITC (1:500) (Cederlane Laboratories, Burlington, Canada) or anti-PAD1 [ ] were used as primary antibodies.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: SUMO chains depolymerization induces slender to stumpy differentiation in T. brucei bloodstream parasites

doi: 10.1101/2023.11.15.567218

Figure Lengend Snippet: Detection of IgM antibodies against VSG221 in the serum of mice infected with wild type (WT) or SUMO chain mutant (SUMO all KR) parasites at the first peak of parasitemia by ELISA. Serum samples were diluted 1:5, and absorbance values (Abs) were measured at 450 nm. C+: positive control; C-: negative control.

Article Snippet: BF cells were collected by centrifugation (1000 x g for 10 min), washed with TDB supplemented with glucose 20 mM and fixed with 4% paraformaldehyde (PFA) in PBS for 1 h. Parasites were allowed to bind to poly-L-lysine coated glass coverslips for 30 min and then incubated with 25 mM NH4Cl for 15 min. Permeabilization and blocking were performed with 3% bovine serum albumin (BSA), 0.5% saponin and 5% normal goat serum in PBS for 1 h. Mouse anti- Tb SUMO (1:500) [ ], mouse anti-VSG221 (1:200), rabbit anti-VSG221 (1:500), mouse anti-EP FITC (1:500) (Cederlane Laboratories, Burlington, Canada) or anti-PAD1 [ ] were used as primary antibodies.

Techniques: Infection, Mutagenesis, Enzyme-linked Immunosorbent Assay, Positive Control, Negative Control

Journal: bioRxiv

Article Title: SUMO chains depolymerization induces slender to stumpy differentiation in T. brucei bloodstream parasites

doi: 10.1101/2023.11.15.567218

Figure Lengend Snippet: WT or SUMO chain mutant (SUMO all KR) parasites were first incubated with anti-VSG221 IgG at 4°C in HMI-9, followed by an incubation at 37°C for 5 and 10 minutes. (A) Parasites were fixed and stained with anti-mouse-Alexa Fluor 488 (green) and DAPI (blue). (B) Whole-cell extracts (corresponding to 1×10 7 cells) were obtained after the 10-minute incubation at 37°C and boiled in Laemmli’s sample buffer. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane, followed by immunoblotting with anti-mouse-Alexa Fluor 790.

Article Snippet: BF cells were collected by centrifugation (1000 x g for 10 min), washed with TDB supplemented with glucose 20 mM and fixed with 4% paraformaldehyde (PFA) in PBS for 1 h. Parasites were allowed to bind to poly-L-lysine coated glass coverslips for 30 min and then incubated with 25 mM NH4Cl for 15 min. Permeabilization and blocking were performed with 3% bovine serum albumin (BSA), 0.5% saponin and 5% normal goat serum in PBS for 1 h. Mouse anti- Tb SUMO (1:500) [ ], mouse anti-VSG221 (1:200), rabbit anti-VSG221 (1:500), mouse anti-EP FITC (1:500) (Cederlane Laboratories, Burlington, Canada) or anti-PAD1 [ ] were used as primary antibodies.

Techniques: Mutagenesis, Incubation, Staining, SDS Page, Membrane, Western Blot

Journal: bioRxiv

Article Title: SUMO chains depolymerization induces slender to stumpy differentiation in T. brucei bloodstream parasites

doi: 10.1101/2023.11.15.567218

Figure Lengend Snippet: (A) Blood smears from the first peak of parasitemia were stained with DAPI for nucleus and kinetoplast configuration analysis (**, p<0.01). (B) Quantification of RNA transcript levels of the characteristic stumpy markers PAD1 and PAD2 was performed by qRT-PCR from RNA samples derived from mice infections. WT and SUMO all KR parasites were isolated at the first peak of the parasitemia. Samples were normalized against 7SL. (C) In vitro bloodstream-to-procyclic differentiation induced by cis-aconitate (CA) was analyzed in SUMO all KR and wild type (WT) parasites. Differentiation to the procyclic form was triggered in SDM-79 medium at 28°C using 6 mM CA. The differentiation process was evaluated following changes in the expression of stage-specific surface markers (VSG221 and procyclin) for 48 h by indirect immunofluorescence. Representative images of anti-VSG221 (red)-anti-EP (green) merged images are shown. One out of five representative experiments is shown. (D) WT + 8-pCPT-cAMP and SUMO all KR + 8-pCPT-cAMP parasites were pre-treated with the cAMP analogue 8-pCPT-cAMP [8-(4-Chlorophenylthio) adenosine 3′,5′-cyclic monophosphate] during 24 h before shifting the temperature and adding CA.

Article Snippet: BF cells were collected by centrifugation (1000 x g for 10 min), washed with TDB supplemented with glucose 20 mM and fixed with 4% paraformaldehyde (PFA) in PBS for 1 h. Parasites were allowed to bind to poly-L-lysine coated glass coverslips for 30 min and then incubated with 25 mM NH4Cl for 15 min. Permeabilization and blocking were performed with 3% bovine serum albumin (BSA), 0.5% saponin and 5% normal goat serum in PBS for 1 h. Mouse anti- Tb SUMO (1:500) [ ], mouse anti-VSG221 (1:200), rabbit anti-VSG221 (1:500), mouse anti-EP FITC (1:500) (Cederlane Laboratories, Burlington, Canada) or anti-PAD1 [ ] were used as primary antibodies.

Techniques: Staining, Quantitative RT-PCR, Derivative Assay, Isolation, In Vitro, Expressing, Immunofluorescence