Journal: iScience

Article Title: Downstream transcription promotes human recurrent CNV associated AT-rich sequence mediated genome rearrangements in yeast

doi: 10.1016/j.isci.2024.111508

Figure Lengend Snippet:

Article Snippet: The yeasts treated with 0 mM of H 2 O 2 were also spotted on the plates containing 20 or 30 μg/mL or Zeiocin (HY-K1053, MCE) to test the sensitivity of yeast to the Zeiocin induced DSBs.

Techniques: Recombinant, Selection, Sterility, Marker, DNA Purification, Clone Assay, Magnetic Beads, SYBR Green Assay, DNA Extraction, Sequencing, Software

Journal: Molecular and biochemical parasitology

Article Title: How schistosomes alter the human serum proteome

doi: 10.1016/j.molbiopara.2016.12.007

Figure Lengend Snippet: Identities of the protein spots 1–20 depicted in figure 1 , representing major differences in the normal human serum proteome following exposure to adult schistosomes for 1 hour at 37°C versus control. Yellow highlights proteins known or implicated as being involved in the complement system. The theoretical molecular weight (MW) of each protein is listed as is the observed MW (kDa); pI, theoretical isoelectric point. The Accession Designation for each identified protein is given. The protein “Identification Score” is calculated by Mascot software and the value in parentheses, the “Peptide Count”, indicates the number of matching peptides identified by mass spectrometry in each case. The Protein Score Confidence Interval (CI) is listed (%); scores above 95% are significant, a lower score may still represent the correct identification, but requires corroboration. The Ratio represents the difference in spot volume between schistosome-exposed serum (S) and control serum (C) as determined by DeCyder software (see for an example).

Article Snippet: The membrane was then probed overnight at 4°C with one of the following polyclonal antisera: rabbit anti-human C3 (Abcam, ab117244) or goat anti-human C4 (AbD Serotec (BioRad) AHP1753) or goat anti-human factor B (Quidel A311) antiserum, all at 1:1000 dilution.

Techniques: Molecular Weight, Software, Mass Spectrometry

Journal: Molecular and biochemical parasitology

Article Title: How schistosomes alter the human serum proteome

doi: 10.1016/j.molbiopara.2016.12.007

Figure Lengend Snippet: Example of gel spot analysis by DeCyder software. Top, magnified view of a section of the 2D-DIGE gel with spot number 4 indicated (yellow boundary). This spot is identified as complement factor Bb and is more abundant in the parasite-treated serum sample (upper right) compared with control serum (upper left). Bottom, DeCyder representation of spot 4 (yellow boundary) and surrounding proteins. The software calculates spot peak volume to measure comparative abundance.

Article Snippet: The membrane was then probed overnight at 4°C with one of the following polyclonal antisera: rabbit anti-human C3 (Abcam, ab117244) or goat anti-human C4 (AbD Serotec (BioRad) AHP1753) or goat anti-human factor B (Quidel A311) antiserum, all at 1:1000 dilution.

Techniques: Software

Journal: Molecular and biochemical parasitology

Article Title: How schistosomes alter the human serum proteome

doi: 10.1016/j.molbiopara.2016.12.007

Figure Lengend Snippet: Analysis of complement protein factor B by western blotting. Samples obtained following human serum incubation for 1 hour at 37°C either in the presence of schistosome parasites (+) or without parasites (−) taken at 0 or 1 hour (as indicated) were resolved by SDS-PAGE, blotted to PVDF and probed with anti-human factor B antiserum. The arrow indicates factor B and the arrowhead indicates the position of migration of the large fragment of factor B designated Bb. At right is a depiction of factor B (large rectangle) and beneath this the Bb fragment is indicated (smaller grey rectangle).

Article Snippet: The membrane was then probed overnight at 4°C with one of the following polyclonal antisera: rabbit anti-human C3 (Abcam, ab117244) or goat anti-human C4 (AbD Serotec (BioRad) AHP1753) or goat anti-human factor B (Quidel A311) antiserum, all at 1:1000 dilution.

Techniques: Western Blot, Incubation, SDS Page, Migration

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Generation of Multiple Fluid-Phase C3b:Plasma Protein Complexes During Complement Activation. Possible Implications in C3 Glomerulopathies

doi: 10.4049/jimmunol.1302288

Figure Lengend Snippet: Panel A: Factor I-depleted serum and factor H-depleted serum were reconstituted with 0.5 mM Mg2+ and incubated at 37°C for 15 minutes, to allow for complex formation, followed by addition of 10 mM EDTA to stop the reaction. The samples were then reconstituted with either PBS or the purified depleted component (factor I or factor H) and incubated for 16 hours at 37°C. Samples were separated using 8% SDS-PAGE and immunoblotted for DBP, α1PI and α1AG. Panel B: Factor H-depleted serum was reconstituted with 0.5 mM Mg2+ and incubated at 37°C for the indicated times. Serum aliquots were separated using 8% SDS-PAGE and immunoblotted for DBP, α1PI, α1AG and factor B. Panel C: Factor I-depleted serum alone or reconstituted with 25% (0.25X) or 100% (1X) of the plasma concentration of factor I (70 μg/ml) was activated with CVF (416 U/ml) for 15 minutes at 37°C. CVF activated normal human serum was included as a positive control for complex formation. Serum aliquots were separated using 8% SDS-PAGE and immunoblotted for DBP, α1PI and α1AG. Panel D: Factor H-depleted serum was treated with 100 μg/ml of either an irrelevant mouse IgG or an antibody that neutralizes the serine protease domain of factor I for 15 minutes on ice. Serum samples then were incubated with 0.5 mM Mg2+ at 37°C for the indicated times. Immunoblots for DBP and α1PI complex formation are shown. Panel E: Pooled normal human serum and C3 depleted serum were activated with CVF for 15 minutes, while factor I depleted serum was incubated at 37°C for 15 minutes. The samples were separated using 8% SDS-PAGE and immunoblotted for C3.

Article Snippet: Mouse monoclonal anti-human factor I neutralization antibody (#A247) was obtained from Quidel (San Diego, CA).

Techniques: Incubation, Purification, SDS Page, Concentration Assay, Positive Control, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Generation of Multiple Fluid-Phase C3b:Plasma Protein Complexes During Complement Activation. Possible Implications in C3 Glomerulopathies

doi: 10.4049/jimmunol.1302288

Figure Lengend Snippet: Panel A: Purified C3, fD, DBP and 0.5 mM Mg2+ were mixed in the presence or absence of fB at physiological concentrations and incubated at 37°C for either 15 or 30 minutes. Factor I-depleted serum sample incubated at 37°C for 15 minutes in the presence of Mg2+ was included as a positive control. Mixtures were immunoblotted for DBP (for complex formation) and factor B (for complement activation). Panel B: Purified fB, fD, DBP and 0.5 mM Mg2+ were mixed with either native C3 or hydroxylamine treated C3 (C3-NH2OH), at physiological ratios were incubated at 37°C for 15 minutes. Factor I depleted serum sample incubated at 37°C for 15 minutes in the presence of Mg2+ was used as a positive control. Samples were immunoblotted for DBP to assess complex formation and factor B to verify complement activation.

Article Snippet: Mouse monoclonal anti-human factor I neutralization antibody (#A247) was obtained from Quidel (San Diego, CA).

Techniques: Purification, Incubation, Positive Control, Activation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Generation of Multiple Fluid-Phase C3b:Plasma Protein Complexes During Complement Activation. Possible Implications in C3 Glomerulopathies

doi: 10.4049/jimmunol.1302288

Figure Lengend Snippet: Panel A: Purified C3, fB, fD and DBP were mixed with either factor H alone, factor I alone or a combination of factors H and factor I at physiological ratios and then activated with CVF in the presence of 0.5 mM Mg2+ for the indicated times. Samples were immunoblotted for DBP to assess complex formation and factor B to verify complement activation. C3 β-chain was used as a loading control. Panel B: The uncropped DBP immunoblot of the last six lanes of panel A to show intermediate cleavage products. Panel C: Purified C3, fB, fD and α1AG were mixed with either factor I alone or a combination of factors H and factor I at physiological ratios and then activated with CVF in the presence of 0.5 mM Mg2+ for the indicated times. Samples were immunoblotted for α1AG (center panel) or C3d (right panel) to assess complex formation and breakdown. The positions of C3b, iC3b and C3dg complexes with α1AG are indicated. To compare complex degradation in serum to the purified protein system, factor I depleted serum alone, or factor I depleted serum reconstituted with factor I and activated with CVF for 15 minutes, were separated and immunoblotted for α1AG (left panel).

Article Snippet: Mouse monoclonal anti-human factor I neutralization antibody (#A247) was obtained from Quidel (San Diego, CA).

Techniques: Purification, Activation Assay, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Generation of Multiple Fluid-Phase C3b:Plasma Protein Complexes During Complement Activation. Possible Implications in C3 Glomerulopathies

doi: 10.4049/jimmunol.1302288

Figure Lengend Snippet: Panel A: EDTA-plasma from DDD patients (n=6) and C3GN patients (n=6) were separated using 8% SDS-PAGE and immunoblotted for C3, factor B, and α1AG. As a reference for complex size, factor H and factor I depleted serum samples were included. Panel B: EDTA-plasma from DDD patients (n=6) and C3GN patients (n=6) or healthy controls (n=2) were reconstituted with 7.5 mM Mg2+ and incubated at 37°C for 60 minutes. The samples were then separated using 8% SDS-PAGE and immunoblotted for C3, factor B, α1PI and α1AG. Panel C: 1.3 mg/ml of C3 and 7.5 mM Mg2+ were added to the EDTA-plasma from DDD patients (n=6) and C3GN patients (n=6) and incubated at 37°C for either 30 minutes or 60 minutes. As a reference for complex size, factor H and factor I depleted serum samples were included. The samples were separated using 8% SDS-PAGE and immunoblotted for C3, factor B and α1AG.

Article Snippet: Mouse monoclonal anti-human factor I neutralization antibody (#A247) was obtained from Quidel (San Diego, CA).

Techniques: SDS Page, Incubation