Journal: Frontiers in Cellular Neuroscience
Article Title: Early Developmental PMCA2b Expression Protects From Ketamine-Induced Apoptosis and GABA Impairments in Differentiating Hippocampal Progenitor Cells
doi: 10.3389/fncel.2022.890827
Figure Lengend Snippet: The contribution of PMCA isoforms to the rate of Ca 2+ clearance following 30 mM KCl treatment in the presence or absence of ketamine. (A) The expression of Atp2b1 in GFP- or PMCA2b-GFP expressing cells was qualified using real-time PCR. The raw data were normalized to endogenous Gapdh expression and were calculated based on the 2 −Δ ΔCt method to obtain relative fold change. The expression level in GFP control treated with saline was taken as 1, n = 4. (B) PMCA1 protein level was determined with the Western blot. GAPDH was used as a loading control. The representative images are shown. (C) Quantification of PMCA1 protein level following normalization to endogenous Gapdh level, n = 3. AU, arbitrary units. (D) The efficiency of Atp2b1 silencing with siRNA evaluated using real-time PCR and confirmed at the protein level with the Western blot. The reduction in mRNA level was quantified using the 2 −Δ ΔCt method, and the expression in scrambled-treated cells was taken as 1, n = 3. (E) Halftime of signal decay (t 1/2 ) of individual tracings recorded from scrambled or PMCA1 siRNA-treated cells, presented as interleaved box graph centered at the mean. (F) The expression of Atp2b4 in GFP- or PMCA2b-GFP expressing cells, n = 3. (G) Immunoreactivity of PMCA4 determined with the Western blot. (H) Quantification of PMCA4 protein level following normalization to endogenous Gapdh level, n = 3. AU, arbitrary units. (I) Over-expression of PMCA4b-mCherry protein and the targeting of the fusion protein to the plasma membrane visualized by immunocytochemistry. Scale bar 20 μm. (J) Halftime of signal decay (t 1/2 ) of individual tracings recorded from mCherry- or PMCA4b-mCherry-positive cells, presented as interleaved box graph centered at the mean. Here, the mock-transfected control was cells double transfected with GFP and mCherry. * P < 0.05, ** P < 0.01, *** P < 0.001.
Article Snippet: Rabbit polyclonal anti-PMCA1 (Cat. No. PA1-914), rabbit polyclonal anti-PMCA2 (Cat. No. PA1-915), rabbit polyclonal anti-PMCA3 (Cat. No. PA1-916), mouse monoclonal anti-PMCA4 (Cat. No. MA1-914), rabbit polyclonal anti-NMDAR1 (Cat. No. PA3-102), rabbit polyclonal anti-NMDAR2A (Cat. No. A-6473), rabbit polyclonal anti-NMDAR2B (Cat. No. PA3-104), anti-NF68 (Cat. No. MA5-14981), anti-GAP43 (Cat. No. PA5-34943), normal mouse IgG1, normal rabbit IgG as well as secondary antibodies conjugated with alkaline phosphatase were purchased from Thermo Scientific (USA).
Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Over Expression, Immunocytochemistry, Transfection
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