Hrp conjugated goat anti mouse antibodies

Total proteins were extracted from leaves of transgenic orchid or leaves of orchids infiltrated with agrobacterium carrying vector (pk2GW7), overexpression clones of wild-type Pha13 (pPha13-oe), or the respective A20 and/or AN1 mutant clone (pPha13A20m, pPha13AN1m or pPha13A20mAN1m) as previously described [52 (link)] with some modification. The boiled extraction buffer (4 M urea, 5% SDS, 15% glycerol, 100 mM Tris-HCl, pH 8, with freshly added 2 mM phenylmethylsulfonyl fluoride, 2 mg mL-1 and 1X complete protease inhibitor [Roche]) was used to extract the total proteins. The Pha13 or Pha13 A20 and/or AN1 domain mutant protein was analyzed by immunoblotting with antibody against Pha13 followed by HRP conjugated anti-rabbit antibodies (Abcam). The protein level of tubulin (loading control) was analyzed by immunoblotting with antibody against tubulin followed by HRP conjugated goat anti-mouse antibodies (GE Healthcare Life Sciences).

In vitro ubiquitination assays were performed as described [33 (link)] with modification. An amount of 3 μg purified His-tagged recombinant proteins (described above) were used for each ubiquitination reaction. Reactions were incubated at 30°C for 3 hours and analyzed by SDS-PAGE followed by immunoblot analysis. Blots were probed using anti-FLAG antibodies (Sigma) followed by HRP conjugated goat anti-mouse antibodies (GE Healthcare Life Sciences).