Journal: Cells

Article Title: N -Glycosylation of LRP6 by B3GnT2 Promotes Wnt/β-Catenin Signalling

doi: 10.3390/cells12060863

Figure Lengend Snippet: Human B3GnT2 promotes Wnt/β-catenin signalling and acts at the level of LRP6. ( a , b ) TOPFLASH Wnt reporter assays (upper graphs) and Western blots (lower blots) showing that human B3GnT2, but not B3GnT8, modifies LRP6 and promotes Wnt/β-catenin signalling in a dose-dependent manner. Amounts transfected: LRP6 , 20 ng; hB3GnT2 or hB3GnT8 , as indicated. Data represent mean + SD. **** p < 0.0001. n.s., not significant. ( c ) TOPFLASH reporter assay in HEK293T cells in 96-well format. Amounts transfected: LRP6 , 20 ng; β-catenin , 50 ng; hB3GnT2 , 0.5 ng. Data represent mean + SD. ** p < 0.01 ( d ) Western blots of cell lysates from HEK293T cells in 96-well format. Amounts transfected: hLRP6 , 20 ng; mWnt3a , 8 ng; V5-mFz5 or V5-mFz8 , 20 ng; hB3GnT2 , as indicated.

Article Snippet: Human B3GnT2 was cloned from hB3GnT1 ( hB3GnT2 ) human ORF clone (NM_006577, OriGene Technologies, Herfurt, Germany) and human B3GnT8 was cloned from hB3GnT8 ORF clone (NM_198540, OriGene Technologies, Herfurt, Germany) into pCS2 + expression vectors.

Techniques: Western Blot, Transfection, Reporter Assay

Journal: Cells

Article Title: N -Glycosylation of LRP6 by B3GnT2 Promotes Wnt/β-Catenin Signalling

doi: 10.3390/cells12060863

Figure Lengend Snippet: Multiple N-glycan sites of LRP6 are relevant for B3GnT2-mediated LRP6 promotion. ( a ) Evolutionarily conserved N -glycosylation sites. Red: asparagine (N) residues predicted to be N -glycosylated, with indicated amino acid position above. ( b ) Schematic model of LRP6 extracellular domain and its N -glycosylation sites. N -glycosylation sites are indicated as red filled circles: PE1 (N42, N81, N281); PE2 (N433, N486); PE3 (N692, N859, N865, N926); PE4 (N1039). PE, β-propeller/EGF-like structural domain. ( c ) Western blots of lysates from B3GnT2−/− HEK293T cells transfected with indicated hLRP6 constructs (20ng) alone or with hB3GnT2 (2 ng) in 96-well format. ( d , e ) TOPFLASH reporter assay using lysates of B3GnT2−/− HEK293T cells transfected with indicated hLRP6 constructs alone or with hB3GnT2 shown in ( c ) in 96-well format. ( d ) Wnt signalling activity of samples expressing LRP6 alone. Data represent mean ± SD. **** p < 0.0001. ( e ) Wnt signalling activities of samples expressing LRP6 alone shown in ( d ) are set as 1 and activities of samples expressing LRP6 and hB3GnT2 are normalised to the corresponding samples expressing only LRP6. Data represent mean ± SD. *** p < 0.001, **** p < 0.0001.

Article Snippet: Human B3GnT2 was cloned from hB3GnT1 ( hB3GnT2 ) human ORF clone (NM_006577, OriGene Technologies, Herfurt, Germany) and human B3GnT8 was cloned from hB3GnT8 ORF clone (NM_198540, OriGene Technologies, Herfurt, Germany) into pCS2 + expression vectors.

Techniques: Western Blot, Transfection, Construct, Reporter Assay, Activity Assay, Expressing

Journal: Cells

Article Title: N -Glycosylation of LRP6 by B3GnT2 Promotes Wnt/β-Catenin Signalling

doi: 10.3390/cells12060863

Figure Lengend Snippet: Human B3GnT2 promotes cell surface LRP6 and is required for Wnt/β-catenin signalling. ( a ) Schematic overview of cell surface LRP6 assay is shown on left. ( b ) Cell surface luminescence/fluorescence assay for HiBiT-LRP6-mCherry. HEK293T cells in 96-well plates were transfected as indicated with HiBit-hLRP6-mCherry (20 ng), mMesd (5 ng) and hB3Gnt2 (0.5 ng). Cells were washed after 48 h with HBSS and treated with detection reagent constituting LgBiT (1:200), Fumarazine (1:100), HEPES (1:100) FBS (5%) and phenol free DMEM medium. The bioluminescence from reconstituted HiBit/LgBiT (Nluc) was measured using a Caloristar multiplate reader and the ratio of bioluminescence to fluorescence was plotted as shown in the graph. Data represent mean ± SD. ** p < 0.01; *** p < 0.001. ( c ) Confocal microscopy analysis of cell surface polylactosamine using FITC-LEL. Note that the cell surface polylactosamine level of B3GnT2−/− cells is much lower than HEK293T WT cells. ( d ) TOPFLASH reporter assay (upper graph) and Western blot (lower panels) of lysates from HEK293T WT or B3GnT2−/− cells transfected as indicated in 96-well format. Amounts transfected: hLRP6 , 20 ng; hB3GnT2 , as indicated. Note that the deficiency in signalling activity in B3GnT2−/− cells is rescued by re-expression of human B3GnT2. Data represent mean ± SD. *** p < 0.001.

Article Snippet: Human B3GnT2 was cloned from hB3GnT1 ( hB3GnT2 ) human ORF clone (NM_006577, OriGene Technologies, Herfurt, Germany) and human B3GnT8 was cloned from hB3GnT8 ORF clone (NM_198540, OriGene Technologies, Herfurt, Germany) into pCS2 + expression vectors.

Techniques: Fluorescence, Transfection, Confocal Microscopy, Reporter Assay, Western Blot, Activity Assay, Expressing