To test whether the hub domain is intrinsically capable of subunit exchange, we purified the hub domain and monitored colocalization.
To monitor colocalization dynamics of diVCA and filaments on tethered Arp2/3 complexes, we first identified the locations where individual Arp2/3 complexes were stably tethered and integrated the fluorescence intensities at those locations in both emission channels.
The cells were treated with lysosomal protease inhibitors pepstatin A (PepA) and EST (to prevent degradation within autolysosomes) at different time points and autophagy was assessed by monitoring colocalization of LC3 with the lysosomal marker (LysoTracker Red) by confocal microscopy as well as western blotting for LC3 (Fig. 1E H).
We sequentially labeled these two fractions by pulse chase with mKate2- and EGFP-fused FKBP-AP21967 and monitored the colocalization of the two tagged proteins after 15 min. As shown in Fig. S1, sequentially labeled VAMP2 was retrograde trafficked back into the cells, where the labels colocalized very well, suggesting that the two fractions underwent the same retrograde trafficking process.
As shown in Figure 10 figure supplement 1, incubation with anti-DCC antibodies dramatically decreased the binding of ApNetrin-1 to the SN, as assayed using Pearson's Correlation to monitor the colocalization of green ApNetrin-1-dendra2 and red DCC-mCherry.
The fluorescently labeled samples were then mixed, and monitored for colocalization, as before.
For these experiments, after imaging the IRES 80S complexes with Phe-tRNAPhe(Cy5) delivered by eEF1A, the same channel was washed three times with 1X EPB to remove all unbound components, and a fresh mix of pre-incubated eEF2-eEF1A-GTP-Phe-tRNAPhe(Cy5) was delivered to the flowcell prior to a second round of imaging aimed at monitoring the rescue of colocalization by addition of eEF2.
In order to examine the degree of autophagy induction in individually infected cells, we monitored L. monocytogenes colocalization with LC3 in infected BMDMs isolated from transgenic mice expressing LC3-GFP [28].
The intracellular colocalization of the p AA- co-AAm) NPs was monitored using fluorescence confocal microscopy.
The colocalization of endogenously expressed vimentin and transiently expressed SERT was monitored in 5HT-stimulated CHO- YFP-SERT CHO- YFP-SERT CHO- YFP-SERT
