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

Figure 3. Dot‐blot arrays of D‐motifs.

Figure 3

  1. The principle of the phosphorylation enhancement dot‐blot arrays. Protein constructs are immobilized onto a solid‐phase support where phosphorylation takes place. Afterward, the phosphorylated epitopes are detected through standard Western blot procedures using a phosphorylation‐sensitive antibody.
  2. The schematic structure of the artificial substrates utilized in the dot‐blot arrays. All constructs share the same tags, substrate sites, and linkers: Only the docking motif‐containing fragments differ.
  3. A sample dot‐blot array for detecting JNK‐binding docking motifs. This specific array contains 48 of the 70 motifs tested in total, and was incubated against activated JNK1.
  4. Quantitative analysis of the sample dot‐blot array. All intensities are relative to that of the NFAT4 motif (positive control), error bars were derived from three parallel samples on the same membrane and show standard deviation from the mean (N = 3). “+” denotes additional, non‐overlapping motifs tested from the same protein. “m” refers to murine (non‐human) sequence. (The corresponding ERK2 and p38α 48 motif arrays and the 70 motif arrays for all three MAPKs are shown on Appendix Fig S1 or on Fig EV1).