Journal: Science advances

Article Title: Scaffold coupling: ERK activation by trans-phosphorylation across different scaffold protein species.

doi: 10.1126/sciadv.add7969

Figure Lengend Snippet: Fig. 5. Cis- and trans-phosphorylation contribution to KSR1-mediated ERK activation. (A) The role of trans-phosphorylation on BRAF/KSR1 interaction. Coimmu- noprecipitation assay from HEK293T cells cotransfected with the indicated Flag-tagged KSR1 constructs (1.5 μg each), upon EGF stimulation (50 ng/ml, 5 min), or when cotransfected with HRASV12 (1 μg), after 18-hour starvation (−). (B) Impact of trans-phosphorylation on KSR1-mediated ERK dimerization. Performed as in (A), ERK dime- rization was determined by native electrophoresis. Bands corresponding to ERK monomers and dimers are indicated. (C) ERK dimerization in response to KSR1 inacti- vation and/or IQGAP1 depletion. Determined in endogenous KSR1 immunoprecipitates from cells transfected with HRASV12 (+) in the presence (+) or absence (−) of an shRNA against IQGAP1 after 18-hour starvation. Where indicated, cells were treated with APS-2-79 (5 μM, 2 hours). (D) Effects of IQGAP1 down-regulation on KSR1-bound phosphorylated ERK, as determined by coimmunoprecipitation upon anti-KSR1 immunoprecipitation from wt and IQGAP1 shRNA down-regulated (KD) Cal62 and Hth83 cells. Figures show signal intensity relative to the levels in wt cells. (E) ERK transphosphorylation in IQGAP1 MEK–binding mutant. HEK293T cells were transfected with the indicated MYC-tagged IQGAP1 constructs and stimulated with EGF where shown (+). (F) Effects of KSR1 down-regulation on IQGAP1-bound phosphorylated ERK, as determined by coimmunoprecipitation upon anti-IQGAP1 immunoprecipitation from wt and KSR1 knock-out (−/−) MEFs. In all cases, immunoprecipitations were per- formed with a specific antibody (IP) or with preimmune serum (PI). All the results shown are representative of three independent experiments.

Article Snippet: EGF was obtained from Sigma-Aldrich (no. E9644), APS-2-79 was purchased from MedChem Express (no. HY-100627), Vemurafenib (PLX4032) was acquired from Selleck Chemicals (no. S1267), and Staurosporine was obtained from Sigma-Aldrich (no. S5921).

Techniques: Phospho-proteomics, Activation Assay, Construct, Electrophoresis, Transfection, shRNA, Immunoprecipitation, Binding Assay, Mutagenesis, Knock-Out

Journal: Science advances

Article Title: Scaffold coupling: ERK activation by trans-phosphorylation across different scaffold protein species.

doi: 10.1126/sciadv.add7969

Figure Lengend Snippet: Fig. 9. A model for cis- and trans-phosphorylation impact on ERK activation. As demonstrated in KSR1/IQGAP1 complexes in response to HRASV12 signals or EGF stimulation. Trans-phosphorylation via IQGAP1 can bypass the inhibitory effect of APS-2-79 on KSR1-bound ERK activation.

Article Snippet: EGF was obtained from Sigma-Aldrich (no. E9644), APS-2-79 was purchased from MedChem Express (no. HY-100627), Vemurafenib (PLX4032) was acquired from Selleck Chemicals (no. S1267), and Staurosporine was obtained from Sigma-Aldrich (no. S5921).

Techniques: Phospho-proteomics, Activation Assay

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: bioRxiv

Article Title: New three-dimensional preclinical models to understand and treat liver cancers activated for the β-catenin pathway

doi: 10.64898/2026.04.01.715868

Figure Lengend Snippet: (A) Representative HE staining and immunostaining for GS, cleaved caspase-3 and KI67 in Apc ΔHep organoids formed 8 days after plating in the ClinoStar® and treated with WNTinib at 1 µM or DMSO for 5 days. The black arrows indicate nuclei positively stained for KI67. (B) Quantification of cleaved caspase-3 and KI67 positive cells in DMSO- and WNTinib-treated organoids (n = 5). (C) Expression of Noxa , Bcl2l1 , Glul and Axin2 transcripts evaluated by RT-qPCR (n = 5) in WNTinib- and DMSO-treated organoids. (D) Representative hematoxylin–eosin staining and immunostaining for GS, cleaved caspase-3 and KI67 in βcat Δex3 HCC tumouroids formed 8 days after plating in the ClinoStar® and treated with WNTinib at 5 µM or DMSO for 5 days. The black arrows indicate nuclei positively stained for KI67. (E) Quantification of cleaved caspase-3 and KI67 positive cells in DMSO- and WNTinib-treated βcat Δex3 HCC tumouroids (n = 3). (F) Expression of Noxa and Bcl2l1 transcripts evaluated by RT-qPCR (n = 3) in DMSO- and WNTinib-treated βcat Δex3 tumouroids. All bar graphs represent mean values ± SD, with individual dots corresponding to independent samples. Differences between two sample groups were assessed using a Mann–Whitney test.

Article Snippet: After 8 days of culture in the ClinoStar®, organoids and tumouroids were transferred into two ClinoReactors: one containing DMSO as a control condition and the other containing WNTinib (MedChemExpress) at 1 μM for organoids and 5 μM for tumouroids.

Techniques: Staining, Immunostaining, Expressing, Quantitative RT-PCR, MANN-WHITNEY

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