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Supporting Information

The following Supporting information is available for this article:

Fig. S1. Generation of FCM IP (flow cytometry following immunoprecipitation) raw data for carboxyl-modified latex (CML) bead controls and wild-type and mutant samples. Controls: (a) intrinsic fluorescence of a homogeneous population of untreated CML beads. Covalent coupled of IP antibodies to CML beads. (b) Alexa 488 dye on antibody-conjugated CML beads used as a reference setting to gate Alexa 488 beam scattering (13). (c) Alexa 647 dye on antibody-conjugated CML beads used as a reference setting to gate Alexa 647 beam scattering (13). (d) Positive control gating of FCM IP pull-down samples with bait MAP3K1 and target MAP3K1 conjugated Alexa 488 antibody in order to visualize the efficacy of the IP pull-down. (e) Gating of FCM IP pull-down samples with bait RHOA and target RHOA in order to visualize the efficacy of the IP pull-down. Samples were treated with MAP3K1-conjugated CML beads and were probed with Alexa 488 and 647-conjugated MAP3K4 antibody, then gated. (f) wild type (WT), (g) c.634-8, (h) p.Leu189Arg, and (i) p.Leu189Pro (quantification in Fig. 2b). Samples were treated with RHOA-conjugated CML beads and were probed with Alexa 488-conjugated MAP3K1 antibody then gated. (j) WT, (k) c.634-8, (l) p.Leu189Arg and (m) p.Leu189Pro (quantification in Fig. 3b).

Fig. S2. (a) Individual sample intensities of MAP3K1 input prior to pull-down for MAP3K4 for Fig. 2c were quantified from conventional Western blots using the LICOR software 3.0. (b) After controlling for MAP3K1 loading the intensities were further normalized to actin, showing an average twofold increase of MAP3K4 binding in all mutant samples. (c) Reverse IP using RHOA as bait shows increased binding of MAP3K1 to all mutant samples, about 2.5-fold increase compared to WT samples. These results were normalized to histone as a loading control.

Additional Supporting information may be found in the online version of this article.

FilenameFormatSizeDescription
CGE_1834_sm_fs1.pdf511KSupporting info item
CGE_1834_sm_fs2.pdf514KSupporting info item

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