Journal: Cell Discovery

Article Title: Identification of putative novel O-glycosylations in the NK killer receptor Ncr1 essential for its activity

doi: 10.1038/celldisc.2015.36

Figure Lengend Snippet: Ncr1 expressed on primary NK cells carries O-linked glycosylations. ( a ) Western blot (WB) for GAPDH in the whole-cell lysates of purified NK cells derived from WT and Ncr1 gfp/gfp mice before imunoprecipitation. ( b – d ) Ncr1 protein from WT and Ncr1 gfp/gfp mice was immunoprecipitated with the mNcr1.6 mAb, blotted and stained with the mNcr1.5 mAb ( b ), Jacalin (JAC) ( c ) and wheat germ agglutinin (WGA) ( d ) lectins. WB figures were adjusted for better clarity. ( e – g ) Ratio between the GAPDH staining and the mNcr1.5 mAb ( e ) or lectins ( f , g ) staining (quantified by pixel intensity). Values are shown as mean±s.e.m.; * P <0.05. The figures combine three independent experiments.

Article Snippet: For the western blotting with lectins, the various fusion proteins (5 μg) were run on 10% SDS-PAGE gel, transferred to a nitrocellulose membrane (Tamar, Mevaseret Zion, Israel) and blotted with biotin-conjugated lectins (Jacalin, wheat germ agglutinin, Maackia amurensis lectin II and SNA, all from Vector labs, Burlingame, CA, USA) or with biotin-conjugated HA Ig.

Techniques: Western Blot, Purification, Derivative Assay, Immunoprecipitation, Staining

Journal: Cell Discovery

Article Title: Identification of putative novel O-glycosylations in the NK killer receptor Ncr1 essential for its activity

doi: 10.1038/celldisc.2015.36

Figure Lengend Snippet: Thr 222 and Thr 225 are glycosylated. ( a ) Coomassie staining of the various Ncr1 fusion proteins (5 μg) run on 10% SDS-PAGE gel under reducing conditions. ( b – e ) Western blot (WB) performed on the various Ncr1 fusion proteins shown in a . Staining was performed with Jacalin (JAC) ( b ), wheat germ agglutinin (WGA) ( c ), Maackia amurensis lectin II (MAL) ( d ) or Sambucus nigra (SNA) ( e ) lectins. WB and Coomassie figures were adjusted for better clarity. The figures combine at least three independent experiments. ( f – i ) Ratio between the Coomassie staining and the WB staining (quantified by pixel intensity) of the various lectins. Values are shown as mean±s.e.m.; * P <0.05. ( j – n ) HPLC chromatogram of O-linked glycan release from the Ncr1 Ig ( j ), Ncr1 T222A Ig ( k ), Ncr1 T225A Ig ( l ) and Ncr1 T222 225A Ig ( m ) fusion proteins and dextran standards ( n ). The various Ncr1 Ig fusion proteins N-linked glycans were released in solution with PNGase F before O-linked glycan analysis. The data are shown as fluorescence arbitrary units. The chromatogram shown in j is identical to the one shown in Figure 1c albeit presented in a different time scale. The HPLC chromatograms are representatives of two independent runs.

Article Snippet: For the western blotting with lectins, the various fusion proteins (5 μg) were run on 10% SDS-PAGE gel, transferred to a nitrocellulose membrane (Tamar, Mevaseret Zion, Israel) and blotted with biotin-conjugated lectins (Jacalin, wheat germ agglutinin, Maackia amurensis lectin II and SNA, all from Vector labs, Burlingame, CA, USA) or with biotin-conjugated HA Ig.

Techniques: Staining, SDS Page, Western Blot, Fluorescence