wnt3a protein  (R&D Systems)

Journal: Nature communications

Article Title: Senescent cells perturb intestinal stem cell differentiation through Ptk7 induced noncanonical Wnt and YAP signaling.

doi: 10.1038/s41467-022-35487-9

Figure Lengend Snippet: Fig. 4 | Ptk7 acts through noncanonical Wnt signaling. a Schematic repre- sentation of canonical and noncanonical Wnt signaling pathways. While canonical and noncanonical Wnt signaling pathways share components such as FZD and Dvl, other components such as LRP5/6 and β-Catenin are specific for canonical Wnt signaling. The figure was created with BioRender.com. b Representative images of organoids cultured in corresponding conditioned media with DMSO or inhibitors. Scale bar, 200 μm. c Quantification of data from (b). The percentage of cystic organoids in organoids cultured in conditioned media is shown. d Representative images of organoids treated with DMSO (carrier), Wnt3a or Wnt5a. Scale bar, 200 μm. e Quantification of data from (d). The percentage of cystic organoids in organoids cultured in conditioned media is shown. f Representative images of organoids cultured in Wnt5a with DMSO or inhibitors. Scale bar, 200 μm. g Quantification of data from (f). mean ± s.d. percentage of cystic organoids in organoids cultured in conditioned media. h Representative images of organoids

Article Snippet: Proteins used in ELISAs were obtained from R&D Systems: PTK7-Fc (R&D Systems, #9799-TK), biotinylated Wnt3a (R&D Systems, #BT1324), biotinylated Wnt5a (R&D Systems, #BT645).

Techniques: Protein-Protein interactions, Cell Culture

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: In Vitro Analysis of Hedgehog Acyltransferase and Porcupine Fatty Acyltransferase Activities

doi: 10.1007/978-1-4939-9532-5_19

Figure Lengend Snippet: Sequences of the Shh (top) and Wnt3a (bottom) biotinylated peptides

Article Snippet: We used AnaSpec (Fremont, CA) to synthesize C-terminal biotinylated (Biotin–PEG NovaTag TM ), disulfide-bonded Wnt3a WT (MHL KC(S-)KC(S-)HG LSG SC(S-)E VKT C(S-)WW) and C-terminal biotinylated (Biotin–PEG NovaTagTM) disulfide-bonded Wnt3a S209A (MHL KC(S-)K C(S-)HG LAG SC(S-)E VKT C(S-)WW) and then analyzed by HPLC to assess purity to be greater than 95%.

Techniques:

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: In Vitro Analysis of Hedgehog Acyltransferase and Porcupine Fatty Acyltransferase Activities

doi: 10.1007/978-1-4939-9532-5_19

Figure Lengend Snippet: Sequences of the Shh (top) and Wnt3a (bottom) biotinylated peptides

Article Snippet: We used AnaSpec (Fremont, CA) to synthesize C-terminal biotinylated (Biotin–PEG NovaTag TM ), disulfide-bonded Wnt3a WT (MHL KC(S-)KC(S-)HG LSG SC(S-)E VKT C(S-)WW) and C-terminal biotinylated (Biotin–PEG NovaTagTM) disulfide-bonded Wnt3a S209A (MHL KC(S-)K C(S-)HG LAG SC(S-)E VKT C(S-)WW) and then analyzed by HPLC to assess purity to be greater than 95%.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: An in vitro fatty acylation assay reveals a mechanism for Wnt recognition by the acyltransferase Porcupine

doi: 10.1074/jbc.C117.800136

Figure Lengend Snippet: An in vitro assay for PORCN-mediated Wnt fatty acylation. A, 10 μg of P100 membranes from cells expressing the indicated constructs (empty vector, WT PORCN, or PORCN (H341A)) were incubated with 100 μm linear or disulfide-bonded peptides representing WT Wnt (black bars) or Wnt S209A (gray bars), and 167 μm [125I]IC15:1 CoA, in reaction buffer for 1 h at 37 °C. Biotinylated peptides were captured with streptavidin–agarose beads, and [125I]IC15:1 incorporation was determined by γ-counting. The experiment was performed three times in duplicate; error bars indicate S.D. B, amino acid sequence of WT Wnt (S-S) peptide and disulfide bond positions. C, 1 μl of C-terminal biotinylated (Biotin–PEG NovaTagTM), disulfide-bonded Wnt3a WT (MHL KC(S-)K C(S-)HG LSG SC(S-)E VKT C(S-)WW) diluted 1:50 in 0.1% formic acid or diluted in 1:25 in ammonium bicarbonate was incubated with trypsin (1:100 E:S ratio) for 4 h at 37 °C and analyzed by high-resolution LC–MS over a mass range of m/z 300–2000. Shown is zoomed in mass range for the (M+4H)4+ ions at m/z 715–720 of the intact peptide and the (M+2H)2+ ions of the two trypsin digestion products at m/z 558–562 and m/z 634–640. Calcd, calculated. D–G, P100 membranes from cells expressing WT PORCN were incubated with 100 μm WT Wnt (S-S) peptide and [125I]IC15:1 CoA for the indicated time points (D) or in the presence of increasing concentrations of NaCl (E). F, the assay was performed as described in A, with either [125I]IC15:1 CoA or [125I]IC15:1 free fatty acid. G, the assay was performed as in A, with linear or Wnt (S-S) peptide, with Wnt (S-S) peptide treated with 10 mm DTT, or with membranes (mbs) pretreated with 10 mm DTT. Biotinylated peptides were captured with streptavidin–agarose beads, and [125I]IC15:1 incorporation was determined by γ-counting. Panels D–G represent averages from two to three experiments, each performed in duplicate or triplicate; error bars indicate S.D.

Article Snippet: C-terminal biotinylated (Biotin–PEG NovaTag TM ), disulfide-bonded Wnt3a WT (MHL KC(S-)K C(S-)HG LSG SC(S-)E VKT C(S-)WW) (calculated monoisotopic mass (M+H) +1 = 2860.3052, observed monoisotopic mass (M+H) +1 = 2860.2943), and C-terminal biotinylated (Biotin–PEG NovaTag TM ), disulfide-bonded Wnt3a S209A (MHL KC(S-)K C(S-)HG LAG SC(S-)E VKT C(S-)WW) (calculated monoisotopic mass (M+H) +1 = 2844.3103, observed monoisotopic mass (M+H) +1 = 2844.2959) peptides were synthesized by AnaSpec (Fremont, CA), and then analyzed by HPLC to assess purity to be greater than 95% pure.

Techniques: In Vitro, Expressing, Construct, Plasmid Preparation, Incubation, Sequencing, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: An in vitro fatty acylation assay reveals a mechanism for Wnt recognition by the acyltransferase Porcupine

doi: 10.1074/jbc.C117.800136

Figure Lengend Snippet: Characterization of PORCN mutants and inhibitors in vitro. A, P100 membranes from cells expressing the indicated PORCN mutants were incubated with WT Wnt (S-S) peptide and [125I]IC15:1 CoA; [125I]IC15:1 incorporation was determined as described for Fig. 1A. The percentage of activity was normalized to WT PORCN (100%). The experiment was performed two times in duplicate; error bars indicate S.D. B, 10 μg of P100 membranes expressing WT PORCN were pretreated for 20 min with increasing concentrations of LGK974, and radiolabel incorporation into WT Wnt peptide was determined as in A. GraphPad Prism (v6.00 for Mac OS X, GraphPad Software, La Jolla, CA) was used to calculate an IC50 = 12.89 nm. The experiment was performed two times in duplicate; error bars indicate S.D. C, HEK293FT cells transfected with Super TopFlash or Super FOP and pRL-TK were co-cultured with PORCN-transfected L-Wnt3a cells in the presence of increasing concentrations of LGK974 for 24 h. An IC50 = 7.473 nm was calculated using GraphPad Prism. Each data point represents the average of triplicates; error bars indicate S.D.

Article Snippet: C-terminal biotinylated (Biotin–PEG NovaTag TM ), disulfide-bonded Wnt3a WT (MHL KC(S-)K C(S-)HG LSG SC(S-)E VKT C(S-)WW) (calculated monoisotopic mass (M+H) +1 = 2860.3052, observed monoisotopic mass (M+H) +1 = 2860.2943), and C-terminal biotinylated (Biotin–PEG NovaTag TM ), disulfide-bonded Wnt3a S209A (MHL KC(S-)K C(S-)HG LAG SC(S-)E VKT C(S-)WW) (calculated monoisotopic mass (M+H) +1 = 2844.3103, observed monoisotopic mass (M+H) +1 = 2844.2959) peptides were synthesized by AnaSpec (Fremont, CA), and then analyzed by HPLC to assess purity to be greater than 95% pure.

Techniques: In Vitro, Expressing, Incubation, Activity Assay, Software, Transfection, Cell Culture