Journal: Nucleic Acids Research

Article Title: Functional transitions of the Aspergillus fumigatus iron regulator HapX are governed by conserved domains cooperatively binding [2Fe-2S] clusters

doi: 10.1093/nar/gkaf796

Figure Lengend Snippet: In liquid media, growth inhibition by combinatorial mutation of at least CRR-B and -C is more severe in +Fe compared to −Fe and accelerates with increased expression of the corresponding hapX alleles. For each strain, ( A ) pxylP:hapX , ( B ) pxylP:hapX A/B/C/D , ( C ) pxylP:hapX B/C , and ( D ) pxylP:hapX A/B/C/D,464 , 10 4 conidia were inoculated in triplicate into 96-well microplates in 0.1 mL minimal medium reflecting iron limitation (−Fe; in green) and iron sufficiency (+Fe; 10 μM FeSO 4 ; in red) supplemented with 0.1% xylose for moderate and 1% xylose for high expression of the respective hapX allele. Using the oCelloScope (BioSense Solutions, Denmark) and its UniExplorer software (version 14.0), growth was scored hourly at 37 °C from 2 h post-inoculation (graphs start at the 5 h time point as there was very limited growth before that) until 24 h incubation. In the software, the “ fungi filamentous growth module ” was used to assess the hyphal area up to 2.75 × 10 5 μm 2 when the growth measurement reached saturation. The values in the growth curves represent the mean ± standard deviation of biological triplicates. Below the growth curves are examples of microscopic images of the fungal cultures taken automatically by the oCelloScope at two time points, 12 h and 24 h, underlining the scored growth data.

Article Snippet: Therefore, we applied automated microbial live cell imaging and analysis using the oCelloScope (BioSense Solutions ApS, Denmark) measuring growth by determining hyphal area (Fig. ).

Techniques: Inhibition, Mutagenesis, Expressing, Software, Incubation, Standard Deviation

Journal: F1000Research

Article Title: Validation of commercially available sphingosine kinase 2 antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence

doi: 10.12688/f1000research.10336.2

Figure Lengend Snippet: Details of primary and secondary antibodies.

Article Snippet: Blots were probed with Proteintech rabbit polyclonal anti-SK2 antibody ( A–D ) or ECM Biosciences rabbit polyclonal anti-SK2 antibody ( E–H ).

Techniques:

Journal: F1000Research

Article Title: Validation of commercially available sphingosine kinase 2 antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence

doi: 10.12688/f1000research.10336.2

Figure Lengend Snippet: Immunoblot analyses of lysates from HEK293 and HeLa cells treated with scrambled control siRNA (si-Neg) or SK2 siRNA (si-SK2), and lysates from wildtype (WT) or Sphk2 -/- MEFs. An equal amount (40 µg) of total protein from each sample was run in duplicate. After transferring to nitrocellulose and blocking, the membrane was separated and duplicate samples were probed with either ( A ) Proteintech rabbit anti-SK2 antibody or ( B ) ECM Biosciences rabbit anti-SK2 antibody. SK2 membranes were imaged using a 4 min exposure. The expected band size for SK2 is ∼65 kDa. Membranes were re-probed with mouse anti-α-tubulin antibody as a loading control (2 min exposure), which was detected at 55 kDa as expected. Consistent results were observed from 2-3 (HEK293 and MEF) or 3-4 (HeLa) independent experiments for each antibody.

Article Snippet: Blots were probed with Proteintech rabbit polyclonal anti-SK2 antibody ( A–D ) or ECM Biosciences rabbit polyclonal anti-SK2 antibody ( E–H ).

Techniques: Western Blot, Control, Transferring, Blocking Assay, Membrane

Journal: F1000Research

Article Title: Validation of commercially available sphingosine kinase 2 antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence

doi: 10.12688/f1000research.10336.2

Figure Lengend Snippet: SK2 was immunoprecipitated from HEK293 cell lysate using either ( A ) Proteintech rabbit anti-SK2 antibody or ( B ) ECM Biosciences rabbit anti-SK2 antibody. Normal rabbit IgG antibody was used as an isotype control. Immunoprecipitates (and 40 µg lysate input) were subjected to immunoblot analyses and probed with ( A ) Proteintech rabbit anti-SK2 antibody or ( B ) ECM Biosciences rabbit anti-SK2 antibody. Membranes were imaged using a 4 min exposure. Images are representative of three independent experiments for each antibody. ( C ) SK2 was immunoprecipitated from HEK293 cell lysates (of equal protein) that had been treated with scrambled control siRNA (si-Neg) or SK2 siRNA (si-SK2), using ECM Biosciences rabbit anti-SK2 antibody. Immunoprecipitates were subjected to immunoblot analyses and probed with ECM Biosciences rabbit anti-SK2 antibody. Membrane was imaged using a 4 min exposure. Image is representative of three independent experiments. IgG h/c = IgG heavy chain.

Article Snippet: Blots were probed with Proteintech rabbit polyclonal anti-SK2 antibody ( A–D ) or ECM Biosciences rabbit polyclonal anti-SK2 antibody ( E–H ).

Techniques: Immunoprecipitation, Control, Western Blot, Membrane

Journal: F1000Research

Article Title: Validation of commercially available sphingosine kinase 2 antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence

doi: 10.12688/f1000research.10336.2

Figure Lengend Snippet: ( A ) HeLa or ( B ) HEK293 cells were treated with scrambled control siRNA (si-Neg) or SK2 siRNA (si-SK2), and endogenous SK2 (green) was visualised by immunofluorescence staining and confocal microscopy, using Proteintech rabbit anti-SK2 antibody or ECM Biosciences rabbit anti-SK2 antibody. ( C ) Wildtype (WT) or Sphk2 -/- MEFs were seeded, and endogenous SK2 (green) was visualised by immunofluorescence staining and confocal microscopy, using Proteintech rabbit anti-SK2 antibody or ECM Biosciences rabbit anti-SK2 antibody. Nuclei were stained with DAPI (blue). For each cell line, background staining was examined by staining cells (si-Neg or WT cells) with secondary antibody and DAPI only, and collecting images using both 488nm and 405nm lasers (SK2 + DAPI). Images were taken at 40× magnification; scale bars = 10 µm. Images shown are representative of more than 100 cells from each experiment, and these results were consistent over three independent experiments for each cell line.

Article Snippet: Blots were probed with Proteintech rabbit polyclonal anti-SK2 antibody ( A–D ) or ECM Biosciences rabbit polyclonal anti-SK2 antibody ( E–H ).

Techniques: Control, Immunofluorescence, Staining, Confocal Microscopy