Western Blot Analysis of Protein Extracts

Total protein extracts from yeast, Xoo and rice plants and Western blot analyses were performed as previously described [41 (link),54 (link)]. The primary antibodies used were as follows: Anti-OsSERK2 for detection of OsSERK2 [54 (link)], anti-GFP (Santa Cruze Biotech) for detection of EFR::GFP and EFR::XA21::GFP, anti-LexA (Clontech) for detection of LexA-fused proteins expressed in yeast from pLexA, anti-HA (Covance) for detection of HA-tagged proteins expressed in yeast from pB42AD and anti-EF-Tu (Thermo Fisher Scientific, PA5-27512) antibody to detect EF-Tu in Xoo protein preparations. The appropriate secondary antibody, anti-mouse (Santa Cruz Biotech) and anti-rabbit (GE Healthcare) coupled to horseradish peroxidase were used in combination with chemiluminescence substrates (Thermo) to detect proteins by exposure to film.

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Schwessinger B., Bahar O., Thomas N., Holton N., Nekrasov V., Ruan D., Canlas P.E., Daudi A., Petzold C.J., Singan V.R., Kuo R., Chovatia M., Daum C., Heazlewood J.L., Zipfel C, & Ronald P.C. (2015). Transgenic Expression of the Dicotyledonous Pattern Recognition Receptor EFR in Rice Leads to Ligand-Dependent Activation of Defense Responses. PLoS Pathogens, 11(3), e1004809.

Publication 2015

anti-LexA anti-HA horseradish peroxidase chemiluminescence substrates

Antibodies Antibody Chemiluminescence Ef tu Horseradish peroxidase Mouse Proteins Rabbit Rice plants Western blot analyses Yeast

Corresponding Organization : Joint Genome Institute

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Variable analysis

independent variables

Total protein extracts from yeast
Xoo
Rice plants

dependent variables

Protein expression detected by Western blot analyses

control variables

Experimental conditions and procedures as previously described in references 41 and 54

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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