raf rbd protein gst beads  (Cytoskeleton Inc)

Journal: Nature Communications

Article Title: SHANK3 depletion leads to ERK signalling overdose and cell death in KRAS-mutant cancers

doi: 10.1038/s41467-024-52326-1

Figure Lengend Snippet: a Representative images of GFP-SHANK3 and mCherry-KRASG12V localisation in A549 cells (maximum projections shown; one experiment with this cell line). Insets and yellow arrows indicate colocalization of GFP-SHANK3 with mCherry-KRASG12V at membrane protrusions. b KRAS–SHANK3 SPN-ARR in an open conformation modelled by aligning the RBD and SPN domains of RAF and SHANK3, respectively, on a membrane composed of POPC (1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine)/ Phosphatidylinositol 4,5-bisphosphate/ Cholesterol. c Structural alignment between KRAS–SHANK3 SPN-ARR (model) and nanodisc-bound KRAS–RAF complex (PDB:6PTW). d Analysis of RAF-RBD–KRAS binding in the presence of the SHANK3 SPN domain using the depicted pulldown assay. Samples were resolved on SDS-PAGE gel and stained with Coomassie. A representative gel is shown (three independent experiments). e Quantification of relative FRET efficiency between GFP-KRASG12V (FRET donor) and mRFP-RAF-RBD (FRET acceptor) in siCTRL or siSHANK3 (smartpool SHANK3 siRNA) HEK293 cells. Shown are the individual data points [mean ± s.d., n = 79 (siCTRL) or 87 (siSHANK3) from three independent experiments. Unpaired two-tailed Student’s t -test with Welch’s correction]. f A representative immunoblot and quantification of ERK activation levels (phospho-ERK1/2 (Thr202/Y204) / total ERK relative to loading) in HCT-116 cells expressing GFP-SHANK3 SPN WT or GFP-SHANK3 SPN R12E/K22D (data represent the individual values; mean ± s.d.; mean of control is set to 1.0 by definition; three independent experiments; Kruskal-Wallis one-way ANOVA and Dunn’s post hoc test). g Representative confocal images (middle plane) and quantification of nuclear ERK (indicating ERK activity) in MIA PaCa-2 cells. Yellow arrowheads point to representative nuclei. N/C, nuclear to cytoplasmic ratio. Shown are the individual data points and the population average of each biological replicate (mean ± s.d.; three independent experiments; one-way ANOVA with Holm-Sidak’s multiple comparison test). h Representative images and quantification of tumour growth of HCT-116 cells, transiently expressing GFP-SHANK3 SPN WT or GFP-SHANK3 SPN R12E/K22D, on CAMs. Tumours are delimited by the yellow circles [mean ± s.d.; n = 21 (GFP, SPN WT) or 19 (SPN R12E/K22D) tumours from two independent experiments; Kruskal-Wallis one-way ANOVA and Dunn’s post hoc test]. Source data are provided as a Source Data file.

Article Snippet: The KRAS pulldown assays were done by incubating the indicated recombinant proteins with RAF-RBD glutathione affinity beads from the Ras Pull-Down Activation Assay Biochem Kit (Cat. no. BK008, Cytoskeleton) according to the manufacturer’s instructions.

Techniques: Membrane, Binding Assay, SDS Page, Staining, Two Tailed Test, Western Blot, Activation Assay, Expressing, Control, Activity Assay, Comparison

Journal: JCI Insight

Article Title: NOGOB receptor–mediated RAS signaling pathway is a target for suppressing proliferating hemangioma

doi: 10.1172/jci.insight.142299

Figure Lengend Snippet: ( A ) NGBR depletion decreased the protein levels of NGBR, HRAS, and KRAS in the fraction of biotinylated cell surface proteins. HemSCs surface proteins were biotinylated under nonpermeabilized conditions and isolated using streptavidin agarose resin from the Pierce Cell Surface Protein Isolation Kit as described in Methods. Proteins were determined by Western blot analysis using antibodies that detect endogenous proteins. Pan-cadherin, calnexin, and GS28 are markers of plasma membrane, ER membrane, and Golgi membrane markers, respectively. The plus symbol (+) denotes results for cells treated with the Sulfo-NHS-SS-Biotin reagent; the minus symbol (−) denotes results for cells that were not treated with the biotin reagent but were otherwise used in the kit procedure. The lanes designated “F” show proteins that flowed through the columns because they did not bind the avidin agarose resin, and the lanes designated “E” show proteins that were eluted from the columns after binding to the avidin agarose resin. ( B–D ) NGBR knockdown decreased the FGF2-induced ( B ), VEGF-induced ( C ), and EGF-induced ( D ) activation of HRAS and KRAS in HemSCs. The activated RAS proteins were isolated using GST-RBD beads, and protein levels were determined by Western blot. Data are validated in 3 independent experiments. NGBR, NOGOB receptor; HemSCs, hemangioma stem cells; GST-RBD, GST-tagged Ras-binding domain.

Article Snippet: RAS activity was assessed using GST-RAF1-RBD beads (catalog RF02, Cytoskeleton) according to the manufacturer’s protocol.

Techniques: Isolation, Western Blot, Avidin-Biotin Assay, Binding Assay, Activation Assay