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. 2022 Dec 12;24(2):e55472. doi: 10.15252/embr.202255472

Figure 2. p38 MAPK‐dependent phosphorylation of TFEB‐S401.

Figure 2

  1. Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB‐WT‐FLAG incubated with the indicated kinase inhibitors for 1 h prior to the addition of 200 μM NaAsO2 for 1 h.
  2. Quantification of immunoblot data shown in (A). Data are presented as mean ± SD using one‐way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant, and ****P < 0.0001 from three independent experiments.
  3. Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB‐WT‐FLAG incubated with either 200 μM NaAsO2 for 1 h, EBSS for 4 h, 100 ng/ml EGF for the indicated times or 37 μM Anisomycin for 1 h. Before the addition of EGF or Anisomycin, cells were serum starved for 8 h.
  4. Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB‐WT‐FLAG exposed to 30 J/m2 of UV‐C and allowed to recover in complete medium for the indicated times. Cells were incubated with p38 MAPK inhibitor (20 μM, SB203580) for 1 h before UV‐C irradiation and allowed to recover for 30 min in the presence of the inhibitor.
  5. Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB‐WT‐FLAG depleted of either p38 MAPK (α + β) or JNK1 or JNK2 or JNK(1 + 2) and incubated with 200 μM NaAsO2 for 1 h. Immunoblots are representative of at least three independent experiments.
  6. Immunoblot analysis of in vitro p38 MAPK kinase assay using GST‐TFEB‐PRD as substrate in the presence or absence of either recombinant human active p38α MAPK or ATP.
  7. Quantification of immunoblot data shown in (F). Data are presented as mean ± SD using one‐way ANOVA (unpaired) followed by Tukey's multiple comparisons test, *P < 0.05 from three independent experiments.

Data information: n = 3 biological replicates (each dot represents a biological replicate).

Source data are available online for this figure.