DNA and RNA transfection
Cellular Mosaic: siRNA-mediated knockdown and plasmid transfection​
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Western blot validation confirmed efficient CHC knockdown in U2OS cells, showing strong reduction of clathrin heavy chain protein compared to controls.​​
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A mosaic culture approach was used, where knockdown and control cells were analyzed side by side within the same dish, ensuring direct comparison under identical experimental conditions.
Confocal imaging of fibril-treated mosaic cultures showed that cells lacking CHC internalized α-synuclein fibrils just as efficiently as neighboring control cells. The fibrils rapidly trafficked to lysosomes regardless of CHC expression.
Generation of LAMP1-RFP stable cell lines
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Since PFFs tagged with Alexa488 are sensitive to cellular permeabilization (reduces PFF fluorescence), I generated multiple LAMP1-RFP expressing cell lines. This was done through transfection of the LAMP1-RFP plasmid and selection of stably expressing colonies using Puromycin. ​​