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. 2015 Nov 4;11(11):837. doi: 10.15252/msb.20156269

Figure 4. Bimolecular fragment complementation (BiFC) experiments.

Figure 4

  1. The principle of YFP fragment complementation driven by MAPK–partner protein interaction. The weak and transient interactions between MAPKs and its binding partners still lead to well‐detectable signals.
  2. Summary of the BiFC experiments. In addition to successfully testing six novel docking motif‐dependent interactions, three positive controls (MKK1, MKK6, and JIP1) and an extra negative control (FAM122A) were also introduced into this analysis. Red squares indicate positive BiFC results (which were mostly directly predictable from fragment‐based experiments). However, some interactions suggested by dot‐blot experiments and/or FP titrations were not seen in BiFC (lined squares). These were possibly too weak or absent in the cellular context.
  3. Bright‐field image of transiently co‐transfected HEK293 cells overlaid with the fluorescence image. Although expression levels and complementation efficiency vary between cells, ablation of D‐motifs results in robust fluorescence intensity changes for known MAPK–partner protein pairs (MKK1‐ERK2, MKK6‐p38α, and JNK1‐JIP1, from left to right, upper panels) similar to a novel MAPK partner (AAKG2, lower panels).
  4. Results of fluorescence measurements on co‐transfected cell populations with positive controls and for AAKG2. (Error bars show standard deviations from the mean, N = 6). Similar expression levels of FLAG‐tagged proteins (wild‐type or D‐motif lacking versions of known or putative MAPK partners) or MAPKs were confirmed by Western blotting using anti‐FLAG (ERK2 and JNK1) or anti‐p38α antibodies. Further BiFC results are shown on Appendix Fig S4.