Quantifying Phosphorylation of Kap1 in nHDF Cells

A total of 4 × 10⁴ nHDF cells were seeded on gelatin-coated coverslips and analyzed by immunofluorescence (36 (link)) using the following antibodies: anti-MCPyV LT (Cm2B4; Santa Cruz) (1:500), anti-Kap1 (catalog no. 10483; Abcam) (1:1,000), anti-pKap1 S824 (catalog no. 70369; Abcam) (1:1,000), anti-mouse Alexa Fluor 488 (Life Technologies; catalog no. A-11001), anti-mouse Cy5 (A10523), and anti-rabbit Alexa Fluor 555 (A-21428). Staining was analyzed on a Nikon spinning-disk confocal microscope with a 20× nonconfocal lens objective or a 100× confocal lens objective. Quantification of phosphorylation intensities was performed by applying the Fiji software package and ImageJ, evaluating 100 cells each.

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Siebels S., Czech-Sioli M., Spohn M., Schmidt C., Theiss J., Indenbirken D., Günther T., Grundhoff A, & Fischer N. (2020). Merkel Cell Polyomavirus DNA Replication Induces Senescence in Human Dermal Fibroblasts in a Kap1/Trim28-Dependent Manner. mBio, 11(2), e00142-20.

Publication 2020

anti-MCPyV LT (Cm2B4; anti-Kap1 anti-pKap1 S824 mouse Alexa Fluor 488 Alexa Fluor 555 spinning-disk confocal microscope

Alexa fluor 488 Alexa fluor 555 Antibodies Cells Confocal microscope Gelatin Immunofluorescence Kap1 Lens Mcpyv Mouse Phosphorylation Rabbit

Corresponding Organization :

Other organizations : University Medical Center Hamburg-Eppendorf, Universität Hamburg, Leibniz Institute of Virology (LIV)

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Variable analysis

independent variables

Seeding of nHDF cells on gelatin-coated coverslips

dependent variables

Immunofluorescence staining of cells using the following antibodies:
- anti-MCPyV LT (Cm2B4; Santa Cruz) (1:500)
- anti-Kap1 (catalog no. 10483; Abcam) (1:1,000)
- anti-pKap1 S824 (catalog no. 70369; Abcam) (1:1,000)
- anti-mouse Alexa Fluor 488 (Life Technologies; catalog no. A-11001)
- anti-mouse Cy5 (A10523)
- anti-rabbit Alexa Fluor 555 (A-21428)
Quantification of phosphorylation intensities

control variables

Total of 4 × 10^4 nHDF cells seeded
Gelatin-coated coverslips
Staining analyzed on a Nikon spinning-disk confocal microscope with a 20× nonconfocal lens objective or a 100× confocal lens objective
Quantification of phosphorylation intensities performed by applying the Fiji software package and ImageJ, evaluating 100 cells each

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