Video 4. Colocalization of FMNL1 and anti-phospho-Ser PKC substrate at the immune synapse (IS) interface.
Untransfected C3 cells (Control YFP-) (first left column) or C3 cells transfected with either the bi-cistronic, YFP expression plasmid interfering all FMNL1 isoforms (shFMNL1-HA-YFP) (second column), or FMNL1 interfering and re-expressing interference-resistant YFP-FMNL1βWT (shFMNL1-HA-YFP-FMNL1βWT) (third column) constructs were mixed with CMAC-labeled, SEE-pulsed Raji cells (blue) attached to slides, as described in Materials and methods. After 1 hr of culture, synaptic conjugates were fixed and labeled with anti-FMNL1 (red) and anti-Phospho-Ser PKC substrate (magenta, changed to blue). YFP fluorescence was in yellow (changed to green). Upper panels, the video (10 fps) shows fixed whole cells (top row) and synapse contact areas (second row) that were imaged with confocal microscopy and Z stacks were processed to generate en face, zx views of the synaptic interface (final frame in the video), as indicated in Materials and methods. Colocalization pixels (white) of FMNL1 (red) with anti-phospho-Ser PKC substrate (blue) are shown. Lower panels, same as upper panels, but the top views and the IS interfaces were generated and stained with anti-phospho-Ser PKC substrate (blue) merged to YFP fluorescence (green). This video is related to Figure 8—figure supplement 1.