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. 2022 Apr 4;14(5):e14649. doi: 10.15252/emmm.202114649

Figure 2. ∆N‐DGKk localizes and impacts cellular signaling similarly to full‐length protein.

Figure 2

  1. HeLa cells transfected with indicated plasmids expressing mouse (m) or human (h) DGKk were analyzed by immunofluorescence confocal microscopy for HA tag (red) and Dapi staining (blue). ∆C1‐hDGKk construct bears deletion of phorbol ester C1 domain (328–449).
  2. Immunoblots and quantification of lysates from Cos‐1 cells transfected with plasmid pCI‐HA‐∆N‐DGKk, untransfected (NT), or plasmid pCI control, with the indicated amount of plasmid (µg) and after 24 h, incubated with puromycin (20 µg/ml, 30 min) to measure rates of protein synthesis. GAPDH was used as a loading control. 0 indicates no puromycin treatment, −/+ indicates treatment without or with DGK inhibitor (DGKi) 3 µM R59022 and 0.2 µM R59949 at 6 µM, 15 min. Densitogram of puromycin incorporation is presented as change relative to mock transfected conditions. Each point represents data from an individual culture, and all values are shown as mean ± SEM. P values were calculated using one‐way ANOVA with Tukey’s multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001.
  3. Representative immunofluorescence staining of cortical neuron cultures transduced at 8 DIV (days in vitro) with 10e9 VG/ml of AAVRh10‐GFP or AAVRh10‐∆N‐DGKk and assessed after 5 days using anti‐MAP2 and anti‐HA for ∆N‐DGKk or direct 488 nm excitation for GFP. DAPI was used to visualize nuclei on merged images. Scale bar, 40 µm.
  4. Representative immunoblots of lysates from WT and Fmr1‐KO cortical neurons transduced with AAVRh10‐∆N‐DGKk (AAV ∆N‐DGKk), at the indicated titers (VG/ml culture volume) and quantification of phosphorylation and total levels of eIF4E. GAPDH was used as a loading control. For quantification, the phospho‐protein signal was normalized first against total protein signal and is presented relative to the signal for WT culture. Each point represents data from an individual culture, and all values are shown as mean ± SEM. P values were calculated using one‐way ANOVA with Tukey's multiple comparison test. **P < 0.01.

Source data are available online for this figure.