corningtm biocoattm fibronectin coated coverslips  (Fisher Scientific)

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: To visualize intracellular NHE6 and CDK5 localization, AP‐1 cells stably expressing NHE6 HA were plated on 18‐mm fibronectin‐coated glass coverslips (#64‐0714; Warner Instruments) and transfected with 0.5 μg CDK5 FLAG DNA the following day.

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: 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: SH‐SY5Y cells were plated on 12 mm fibronectin‐coated coverslips (1254580; Fisher Scientific) and allowed to grow for 2–3 days.

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