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. 2021 Dec 23;23(3):e53135. doi: 10.15252/embr.202153135

Figure 6. Domain A interferes with an interaction of STIM1 with PDE8B and increases NFAT translocation.

Figure 6

  1. Mean LFQ intensities of interaction candidates of three replicates each with pull‐down experiments of HA‐tagged STIM1A‐mCherry and STIM1‐mCherry after expression and TG activation in HEKS1/S2−/− cells. Eluates were analyzed via mass spectrometry, and only hits with significant differential scores (P < 0.05, Student’s t‐test) are shown.
  2. Hits from (A) tested for interaction with commercially available constructs quantified with bimolecular fluorescence complementation (BiFC) via flow cytometry. HEKS1/S2−/− were transfected with STIM1‐YFPC (black) or STIM1A‐YFPC (red) in combination with POI‐YFPN and screened for YFP+ cells via FACS; results (mean ± SD) were obtained from three transfections each with 10,000 sorted cells.
  3. Correlation of the mean LFQ intensities from (A) and YFP+ cells from BiFC assay from (B). Dotted lines show differences in interaction of the POIs with STIM1 in comparison with STIM1A.
  4. Ratio of nuclear NFAT‐GFP vs. cytosolic GFP intensity normalized to t = 0 (mean ± s.e.m.) after transfection of SH‐SY5Y S1−/− cells with STIM (n = 42), STIM1A (n = 82), STIM1A_D503A (n = 49), or vector‐only (ØSTIM, n = 15) IRES‐mCherry and induction of SOCE after stimulation with 1 µM TG. Data (total n as indicated) were obtained from at least three independent transfections.
  5. Normalized endpoint ratios at 30 min (mean ± s.e.m.) from numbers indicated in (D). *P < 0.05, Kruskal–Wallis ANOVA with Dunn’s multiple comparisons test.