Journal: eLife

Article Title: Selective activation of FZD7 promotes mesendodermal differentiation of human pluripotent stem cells

doi: 10.7554/eLife.63060

Figure Lengend Snippet: ( A ) Schematic of FZD7- and LRP6-specific binders. Red blocks depict the variable light (V L ) and heavy (V H ) antibody domains that recognize FZD7 and were fused to form the single-chain variable fragment (scFv) in F7 and F7L6. Purple blocks depict the scFv that recognizes LRP6 and is used in L6 and F7L6. ( B ) Expression of FZD7- and LRP-specific binders. Transgenes encoding F7-Ab, F7, L6, and F7L6 were stably transduced in CHO cells. Recombinant proteins were harvested and purified from conditioned media and detected by Coomassie Blue staining (upper) and anti-human-Fc immunoblot (lower). ( C ) Binding specificity of F7, L6, and F7L6. HEK293T carrying mutations in FZD1, 2, and 7 (F127-KO) or in LRP6 (LRP6-KO) were transfected with FZD7 and LRP6, respectively, and whole cell lysates were probed in a dot blot format with conditioned media containing F7, L6, or F7L6. As a negative control (Mock), blots were incubated with CM from untransfected CHO cells. ( D ) Schematic of FZD7 and amino acid alignment of the extracellular ‘neck’ region of hFZD7 and mFzd7. The dashed oval indicates the neck region. The red box in the amino acid sequences indicates amino acid position 188. ( E ) HEK293T cells were transiently transfected with the indicated V5-tagged transgenes, and cell lysates were probed with either F7-Ab or V5 antibody (α-V5). Mock = untransfected cells. ( F ) Mapping F7-Ab epitope to an eight amino acid sequence. Bacterial lysates containing fusion proteins between GST and the FZD7 peptide sequences indicated in the table were detected by Coomassie staining (top) or by immunoblotting with F7-Ab (bottom). Abbreviations in table: aa188, amino acid at position 188; aa#, number of amino acids in FZD7 peptide; L, leucine; P, proline. ( G ) F7-Ab is specific to human FZD7 and does not cross-react with the other nine FZDs (1-6, 8-10). F127-KO were transfected with expression constructs carrying the indicated human FZD cDNAs tagged with an intracellular V5 sequence. Non-permeabilized cells were stained with F7-Ab for cell-surface FZD expression, and then permeabilized and stained for V5 expression. All FZD receptors were expressed as revealed by anti-V5 antibody staining.

Article Snippet: Primary antibodies and dilutions used: anti-FZD7 (F7-Ab) (1:1000 of 1 mg/mL stock), V5 tag antibody (GeneTex/GTX628529/1:4,000), monoclonal anti-β-catenin antibody (Sigma-Aldrich/C7207/1:2,000) (RRID_AB_476865), anti-β-actin antibody (Sigma-Aldrich/A2228/1:5,000) (RRID: AB_476697 ).

Techniques: Expressing, Stable Transfection, Recombinant, Purification, Staining, Western Blot, Binding Assay, Transfection, Dot Blot, Negative Control, Incubation, Sequencing, Construct

Journal: eLife

Article Title: Selective activation of FZD7 promotes mesendodermal differentiation of human pluripotent stem cells

doi: 10.7554/eLife.63060

Figure Lengend Snippet:

Article Snippet: Primary antibodies and dilutions used: anti-FZD7 (F7-Ab) (1:1000 of 1 mg/mL stock), V5 tag antibody (GeneTex/GTX628529/1:4,000), monoclonal anti-β-catenin antibody (Sigma-Aldrich/C7207/1:2,000) (RRID_AB_476865), anti-β-actin antibody (Sigma-Aldrich/A2228/1:5,000) (RRID: AB_476697 ).

Techniques: Recombinant, Plasmid Preparation, Western Blot, Immunofluorescence, Control, Purification, Produced, Coomassie Assay, Reverse Transcription, SYBR Green Assay, Staining