Journal: Acta Physiologica (Oxford, England)

Article Title: The Recycling Endosomal (Na + , K + )/H + Exchanger NHE 6/ SLC 9 A 6 Facilitates Signal Transduction by Shuttling Cyclin‐Dependent Kinase 5 to the Plasma Membrane

doi: 10.1111/apha.70230

Figure Lengend Snippet: Immunofluorescence confocal microscopy of NHE6 and CDK5 in AP‐1 and SH‐ SY5Y cells. (A) AP‐1 cells stably expressing NHE6 HA (AP‐1/NHE6 HA ) and (B) SH‐SY5Y cells were grown on 18‐mm fibronectin‐coated glass coverslips. AP‐1/NHE6 HA cells were grown for 1 day, whereas SH‐SY5Y cells were grown for 2–3 days to allow time for neuronal processes to form. Cells were then transfected with 0.5 μg CDK5 FLAG and fixed after 24 h. Cells were stained with αNHE6 antibody (αNHE6 p ) and either monoclonal αFLAG (αFLAG m ) or αCDK5 (αCDK5 m ) antibodies. Cells were imaged by confocal microscopy and are displayed as maximum intensity projections of z‐stacks. (A) Representative image of NHE6 HA (magenta) and CDK5 FLAG (green) localization in AP‐1 cells. Membrane ruffles enriched with both proteins are circled in white. This experiment is representative of two independent trials. Scale bar: 10 μm. (B) Representative image of endogenous NHE6 (magenta) and CDK5 (green) localization in SH‐SY5Y cells with neuronal‐like processes (N‐type). Areas with high colocalization are indicated with white arrows, including a zoomed‐in image of a neuronal process in the merged image. Two independent trials were performed. Scale bar: 10 μm. (C, D) Manders' colocalization coefficients (MCCs, M1, and M2) were calculated using ImageJ analysis software for NHE6 and CDK5 in individual transfected AP‐1 cells ( n = 6) (C) and SH‐SY5Y neurons ( n = 22) (D) from two independent experiments. The bar graph shows the mean ± SD. Statistical analysis was performed using a paired Student's t ‐test (** p < 0.01, *** p < 0.001).

Article Snippet: The number of PLA signals per cell was quantified using Imaris image analysis software.

Techniques: Immunofluorescence, Confocal Microscopy, Stable Transfection, Expressing, Transfection, Staining, Membrane, Software

Journal: Acta Physiologica (Oxford, England)

Article Title: The Recycling Endosomal (Na + , K + )/H + Exchanger NHE 6/ SLC 9 A 6 Facilitates Signal Transduction by Shuttling Cyclin‐Dependent Kinase 5 to the Plasma Membrane

doi: 10.1111/apha.70230

Figure Lengend Snippet: Colocalization of NHE6 and CDK5 in SH‐SY5Y cells using the proximity ligation assay. (A) SH‐SY5Y cells were grown for 2–3 days prior to fixation and permeabilization. Cells were incubated without a primary antibody, with αNHE6 p or αCDK5 m individually or both primary antibodies overnight and washed thoroughly. Next, samples were incubated with secondary proximity ligation assay (PLA) probes. After extensive washing, samples were incubated with a solution containing DNA ligase and connector oligonucleotides to ligate PLA probes in close proximity to each other. Finally, samples were incubated with a solution containing DNA polymerase and fluorescently tagged oligonucleotides to amplify and detect the signal. Nuclear DNA was stained with DAPI (4′,6‐diamidino‐2‐phenylindole). Samples were visualized by fluorescence confocal microscopy and representative images are displayed as maximum intensity projections of z‐stacks. White arrowheads point to areas of colocalization at or near the plasma membrane, whereas green arrowheads indicate colocalization within the cell. Images are representative of two independent trials. Scale bar: 10 μm. (B) The number of PLA positive signals per cell were quantified using Imaris image analysis software. Data points represent the number of PLA signals from each individual cell, while the scatter interval plot shows the average number of signals from all the cells ( n = 63–73) in both trials. Values represent the mean ± SD. Statistical analysis was performed using a two‐way ANOVA analysis with post hoc Tukey's tests (*** p < 0.001).

Article Snippet: The number of PLA signals per cell was quantified using Imaris image analysis software.

Techniques: Proximity Ligation Assay, Incubation, Staining, Fluorescence, Confocal Microscopy, Clinical Proteomics, Membrane, Software