Anti rfp

For utricle staining, explants were fixed in 4% paraformaldehyde for one hour and washed with PBS containing 0.1% TritonX-100. Primary antibodies in this study used were anti-Moysin7a (1:500, rabbit; Proteus), anti-Sox2 (1:500, rabbit; Millipore), anti-RFP (1:500, Rabbit; Millipore), anti-GFP (1:500, chicken; Abcam) and anti-CD326 (1:200; rat; Thermo Fisher; Catalog number 17-5791-82). Secondary antibodies used were Alexa Fluor 488 and Alexa Fluor 594 (1:2000, Invitrogen). Cell nuclei were labeled by incubation in 0.5 ug/mL of DAPI in PBS for 20 min. Immunofluorescence images were captured on a Zeiss AxioImager microscope with Apotome structured illumination..

Primary antibodies used in this study were anti-RFP (1:5,000, rabbit; Millipore), anti-Atoh1 (1:50,000, chicken; a gift from Matthew Kelley and Thomas Coate; Driver et al., 2013 (link)) and anti-GAPDH (1:5,000, chicken; Millipore). Experiments were performed under standard Western protocol with Amersham ECL western blotting detection (GE Healthcare). Images were captured by a LAS-4000 Mini luminescent image analyzer.

We harvested infiltrated leaves at 48–72 h after transient Agrobacterium-mediated infiltration and extracted total proteins from frozen, ground tissue by mixing with 3 Laemmli buffer at a 1:1 ratio (w/v) for 5 min. Cell debris was collected by centrifugation at 12,000 g for 5 min at 4°C, after which we determined the protein concentration in the supernatant by Bio-Rad Protein assay. Equal amounts of total protein were aliquoted, brought to 40 μL with protein extraction buffer, and mixed with 10 μL 5× SDS-PAGE loading buffer. After boiling the samples for 10 min at 100°C, we quickly chilled the total protein lysate on ice for 5 min, centrifuged the sample at 12,000 g for 1 min at 4°C, and separated the proteins on SDS-PAGE gels. We performed immunoblotting with anti-myc (Sigma, St. Louis, USA), anti-RFP (Sigma, St. Louis, USA), and anti-GFP (Sigma, St. Louis, USA) primary antibodies and anti-rabbit or anti-mouse (Sigma, St. Louis, USA) secondary antibody at 1:10,000 dilution. Antibodies of TuMV CP, RSV CP and P3 proteins were prepared in our laboratory.
For IP, protein extracts were incubated with anti-GFP antibody for 4 h at 4°C. The beads were washed six times with ice-cold IP buffer at 4°C. The IP samples were analyzed by SDS-PAGE, immunoblotted using anti-myc antibody, and detected using Pierce ECL Western Blotting Substrate (Amersham Image 680).