Using qRT-PCR, immunoblots, co-immunoprecipitation assays, and immunofluorescent labeling, we observed that pRb loss resulted in Rac1 and Pak1 overexpression concomitant with merlin inactivation by Pak1, merlin detachment from the membrane, and adherens junction loss.
By double MECA-79 and CXCL12 immunofluorescent labelling, we observed that MECA-79-positive HEVs preferentially displayed CXCL12 compared with flat MECA-79-negative vessels.
The presence or absence of ORCs and GINs bound to newly replicated chromatin was re-examined after labeling cells with EdU for 30 or 40 min. As expected, with 30-min labeling we observed longer EdU tracks.
However, when the 23 nt (green) strand of the upstream duplex presenting a 3′-hydroxyl end at the junction point was labelled we observed the formation of a new, larger DNA species.
However, due to the UPRT-independent thiol-labeling we observed, it is likely that TU-tagging of rare cell types will always have a signal-to-noise problem to some extent.
By analyzing cell death using Annexin V/7AAD labeling, we observed only a slight increase in cells in early (Annexin V+/7AAD−) and late (Annexin V+/7AAD+) apoptosis after RTX and GA101 treatment.
We were also concerned that the ssDNA labeling we observed might be the result of exonuclease-deficient (exo–) Klenow's strand displacement synthesis extending the 3′-end of a nick in the DNA and displacing the downstream DNA strand rather than the result of filling ssDNA gaps in the template.
In dual color labeling experiments, we observed that Cy5-labeled Gas6 (58 nM) and Dylight488-labeled Protein S (55 nM) bound to the same subpopulation of cells, with no reduction in binding when compared with single label only controls.
Using fluorescent labelled Phalloidin, we observed co-localization of TRPV4 with actin at various actin cytoskeleton enriched regions, such as the actin ribs of the cortical membranous regions, and at filopodial and lamellipodial structures (Fig. S3).
It should be noted, however, that in rats, the CSPG structures appear to surround the subcompartment [44], [45], instead of exhibiting a clear overlap between striosomes and WFA labelling as we observed in the mouse.
