Skip to main content
. 2022 May 19;23(7):e54163. doi: 10.15252/embr.202154163

Figure EV4. Members of the Menorin pathway are N‐glycosylated.

Figure EV4

  1. Schematic of endoglycosidase activity on N‐glycan chains. The gray shading represents parts of the chain that are cleaved by each respective endoglycosidase. Glycan residues are consistent with Fig 2A, and colors of endoglycosidases correspond to Fig 3E and G.
  2. Western blot against GFP in C. elegans lysate DMA‐1::GFP (qyIs369), after precipitating with anti‐GFP antibody. Control indicates an otherwise wild‐type background as opposed to an aman‐2(gk248486) background. The red boxed +F indicates that the lysate is treated with the PNGase F glycosidase, while the green boxed +D corresponds to the Endo D glycosidase, which cleaves paucimannose type N‐glycans. Size shifts indicate that some paucimannose structures are present on DMA‐1 (left), and that the aman‐2 mutant results in the loss of paucimannose structures on DMA‐1 (right). Ladder is marked in kilodaltons (kDa). The experiment was repeated four times with biological replicates.
  3. Western blot against GFP in C. elegans lysate expressing no transgenes (N2) and expressing KPC‐1::GFP (dzEx1865), after precipitating with anti‐GFP antibody. The molecular weights of wild‐type lysate are 130 and 150 kDa. The red boxed plus sign indicates that the lysate is treated with the PNGase F glycosidase. The downward size shift reveals that N‐glycan structures are present on KPC‐1. In the bottom blot, control indicates an otherwise wild‐type background as opposed to an aman‐2(gk248486) background. No visible size shift is observed, and the experiment was repeated three times with unique samples.
  4. Western blot against FLAG in C. elegans lysate expressing no transgenes (N2) and expressing SAX‐7::GFP::3XFLAG (ddIs290). Robust expression precludes the need for immunoprecipitation. The molecular weights of wild‐type lysate are 100, 70, and 65 kDa. The red boxed plus sign indicates that the lysate is treated with the PNGase F glycosidase. The downward size shift reveals that N‐glycan structures are present on SAX‐7. In the bottom blot, control indicates an otherwise wild‐type background as opposed to an aman‐2(gk248486) background. No visible size shift is observed, and the experiment was repeated three times with unique samples. The FLAG epitope contains no N‐glycosylation sites.
  5. Western blot against FLAG in C. elegans lysate expressing no transgenes (N2) and expressing endogenous LECT‐2::mNeonGreen::3XFLAG (dz249). Robust expression precludes the need for immunoprecipitation. The molecular weight of wild‐type lysate is 70 kDa. The red boxed plus sign indicates that the lysate is treated with the PNGase F glycosidase. The small downward size shift reveals that N‐glycan structures are present on LECT‐2. In the bottom blot, control indicates an otherwise wild‐type background as opposed to an aman‐2(gk248486) background. No visible size shift is observed, and the experiment was repeated three times with unique samples.