The integral membrane S-locus receptor kinase of Brassica has serine/threonine kinase activity in a membranous environment and spontaneously forms oligomers in planta -- Data Supplement - Supplemental Data -- Proceedings of the National Academy of Sciences

Supplementary material for Giranton et al., Proc. Natl. Acad. Sci. USA, 10.1073/pnas.070025097

Materials and Methods
Immunocytofluorescence Detection of Recombinant Protein.
Amplified recombinant baculovirus driving the expression of the recombinant SRK₃HA protein was used to infect monolayers of exponentially growing cells adhered to poly-L-lysine (Sigma)-treated coverslips (1.5 ´ 10⁶ cells per 35-mm petri dish, multiplicity of infection = 10). At 36 h postinfection, cells were fixed for 20 min in PBS containing 4% (mass/vol) paraformaldehyde and 10% (vol/vol) FCS (GIBCO/BRL). In some samples, cells were permeabilized by addition of 0.2% (mass/vol) Triton X-100. After five washes, the presence of SRK₃HA protein was detected by incubating the coverslips for 1 h at 37°C with mAb 85-36-71 (1/100) or 12CA5 (5 µg/ml, Boerhinger Mannheim), which recognize the N terminus and the HA epitope, respectively (see Fig. 1A of the main text). After washing, coverslips were incubated with a fluorescein-conjugated secondary antibody (1/100, Amersham Pharmacia) for 30 min at 37°C. Rinsed coverslips were mounted in a fluorescence preservative mix (Citifluor, London) and observed under UV light. Negative controls were performed as follows. Cells that express SRK₃HA were incubated with mAb 85-36-71 plus the antigenic peptide recognized by mAb 85-36-71, and cells expressing non-tagged SRK₃ were incubated with 12CA5. Determination of the Topology of Recombinant SRK in Microsomes.
Microsomes were resuspended at a final concentration of 0.8 mg/ml in digestion buffer (10 mM Tris, pH 7.5/20 mM CaCl₂/27 mM KCl/140 mM NaCl/20 µg/ml trypsine) to which variable concentrations of Triton X-100 had been added and incubated at 22°C for 2 h. After digestion, samples were denatured by addition of SDS/PAGE loading buffer and immediately boiled for 5 min.