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. 2021 Apr 21;40(10):e106188. doi: 10.15252/embj.2020106188

Figure 2. Tpl2[D270A] mutation promotes phagosome maturation independently of MAP kinase activation.

Figure 2

  • A–F
    Experiments were performed using murine BMDMs. (A) Intra‐phagosomal proteolysis in WT, Nfkb1[SSAA] (Nfkb1 SSAA/SSAA), and Nfkb1[SSAA]/Tpl2[D270A] BMDMs were monitored as in Fig 1B (n = 4 wells). (B) Intra‐phagosomal acidification in WT, Nfkb1[SSAA], and Nfkb1[SSAA]/Tpl2[D270A] BMDMs was monitored as in Fig 1C (n = 4 wells). (C) Cell extracts from LPS‐stimulated Nfkb1[SSAA] and Nfkb1[SSAA]/Tpl2[D270A] BMDMs were immunoblotted for the indicated antigens. (D) Intra‐phagosomal proteolysis in WT and inhibitor‐treated BMDMs (n = 4 wells) was monitored as in Fig 1B. BMDMs were pre‐treated with 0.1 μM PD0325901 (10 min) to inhibit MEK1, pre‐treated with 1 μM VX‐745 (1 h) to inhibit p38α, and pre‐treated with 10 μM C34 (1 h) to inhibit TPL‐2. (E). Intra‐phagosomal acidification in WT and inhibitor‐treated BMDMs (see (D) for conditions) was assayed as in Fig 1C (n = 4 wells). (F) Cell extracts from LPS‐stimulated, inhibitor‐treated WT, and Tpl2[D270A] BMDMs were immunoblotted for the indicated antigens. 1 μM bafilomycin A1 was added to inhibit V‐ATPases.
  • G–K
    Experiments were performed using human primary monocyte‐derived macrophages. (G) Intra‐phagosomal proteolysis in human macrophages was monitored following uptake of DQ Green BSA / AF594 latex beads. TPL‐2 catalytic kinase activity was blocked by pre‐treatment with 10 μM C34 for 1 h (n = 4 wells). (H) Intra‐phagosomal acidification in human macrophages was monitored following uptake of BCECF‐coupled latex beads (n = 4 wells). (I) Intra‐phagosomal proteolysis in human macrophages was assayed as in Fig 2G. MAP kinase activity was inhibited by combinatorial pre‐treatment with 0.1 μM PD0325901 (MEK1 inhibition) for 10 min and 1 μM VX‐745 (p38 inhibition) for 1 h (n = 4 wells). (J) Intra‐phagosomal acidification in human macrophages was monitored as in Fig 2H (n = 4 wells). (K) Cell extracts from LPS‐stimulated, inhibitor‐treated primary human macrophages were immunoblotted for p‐ERK1/2, ERK1/2, p‐p38, p38, and HSP90.

Data information: One representative experiment out of three shown. Error bars and shaded areas represent SEM. ****P < 0.0001. Panels (A, B, D, E, G, H, I, J) Paired Mann–Whitney t‐test; all differences relative to WT are ****.