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. Author manuscript; available in PMC: 2015 Feb 3.
Published in final edited form as: Curr Protoc Protein Sci. 2014 Feb 3;75:Unit–12.12.. doi: 10.1002/0471140864.ps1212s75

Figure 4. Western and lectin blot analyses of O-mannosylated forms of Drosophila ExDg-FLAG expressed in vivo.

Figure 4

A, Western blot detection of ExDG expressed in rt-tw double mutants (rt− tw−), rt mutants (rt−), tw mutants (tw−), wildtype background (WT), and backgrounds with ubiquitous ectopic expression of RT (rt+), TW (tw+), or RT-TW co-expression (rt+ tw+). L band represents a highly O-mannosylated glycoform, while S band corresponds to a glycoform without significant O-mannosylation. B, Analysis of ExDg glycosylation by glycosidase treatments. The top panel shows Con A reactivity of purified ExDG after treatments with PNGaseF and α-mannosidase. The S glycoform purified from rt mutant background (left side) loses its Con A reactivity either after the removal of N-linked glycans by PNGaseF or after treatment with α-mannosidase removing α-linked mannose residues, suggesting the absence of O-mannose modifications and efficient removal of oligomannose structures either by trimming N-linked branches with α-mannosidase or by complete elimination of N-linked glycans with PNGaseF. The L glycoform purified from RT-TW co-expression background (right side) retains Con A reactivity after treatment with PNGaseF, α-mannosidase, or both glycosidases, suggesting that L glycoform is O-mannosylated, and that α-mannosidase does not remove O-mannose completely. The bottom panel shows anti-FLAG western blot control corresponding to the lectin blot shown in the top panel. Dashed outline shows the region of the L glycoform on the blots. Figure adapted with permission from (Nakamura et al., 2010b).