Alkaline phosphatase

Cells were lysed, and proteins were separated by SDS-PAGE and transferred to PVDF membrane. After blocking of non-specific binding, immunoblots were incubated with monoclonal antibodies against the N-terminal region (a.a. 1–181) of WT1 protein (6F-H2, Dako Cytomation, Carpinteria, CA), GAPDH (6C5, Millipore, Temecula, CA) and His-tag (Anti-Xpress antibody, Invitrogen, Carlsbad, CA) or polyclonal antibody against the C-terminal region (a.a. 431–450) of WT1 protein (C-19, Santa Cruz Biotechnology), followed by incubation with an anti-mouse or rabbit IgG antibody conjugated with alkaline phosphatase (Santa Cruz Biotechnology), and visualized using BCIP/NBT kit (Nacalai Tesque, Kyoto, Japan).

An N‐terminal‐specific peptide of PtrHB4 (SKDKHMDSSKYVRY) was synthesized and injected into rabbits to raise antibodies (Abmart, Shanghai, China) (Figure S1b). Rabbit serum was collected and purified using protein‐A/G Sepharose. The purified antibodies were diluted into a concentration of 1 μg/μL for later use. The N‐terminal 150 amino acids length of PtrHB4 protein was cloned into pET28 vector and then expressed in E. coli. (BL21). Total proteins of cell lysate after IPTG induction were separated by SDS‐PAGE to examination expression of protein (Figure S1c). Western blot was performed using total proteins of cell lysate and from shoot tip of wild type plants against PtrHB4 antibody (diluted at 1 : 1000) to analysis antibody specificity according to the previous protocol (Figure S1d and e) (Song et al., 2010). The secondary antibodies (linked with alkaline phosphatase, Santa Cruz, CA) were diluted in 1 : 5000. The shoot tip and internodes of wild plants were embedded and sliced into 10‐μm‐thick sections for immunolocalization according to the previous protocol (Song et al., 2010). The first antibodies were diluted in 1 : 200. The secondary antibodies were diluted in 1 : 1000. After colour development, the sections were gradually dehydrated with alcohols, cleared with xylene and observed under an OLYMPUS BX51 light microscope (Olympus, NY).