To follow up on the dual mode of SHARP protein function in more detail, we used mammalian two-hybrid assays (Figure S7) and monitored the subcellular localization of NPAS2 upon co-transfection with either BMAL1, SHARP-1 or SHARP-2 encoding expression plasmids (Figure S8).
For this, we monitored the subcellular localization of Hsp104, an aggregate-remodeling chaperone, in wild-type and ncs2Δelp6Δ cells upon diamide exposure.
First, we used immunoblotting to monitor the subcellular distribution of Rab26 during the purification of synaptic vesicles from the rat brain.
To this end, we stained asynchronously growing HeLa cells with specific antibodies, and monitored the subcellular localization of the proteins in interphase and M phase.
To further expand on the role of STK38 in autophagosome formation, we monitored the subcellular localization of ATG14L, WIPI-1, and ATG12.
We monitored the subcellular distribution of the EYFP-Bad and EYFP-BadS136A fusion constructs by epifluorescent microscopy with and without co-transfected Akt-1 and could not detect altereded subcellular localizations (data not shown).
Importantly, MarsCy1 fusion did not perturb normal Lgr5 trafficking (Additional file 3: Figure S3 and Additional file 4: Figure S4) and could be used in combination with SCi1 to monitor the subcellular distribution of Lgr5 and its internalization by confocal microscopy (Fig. 1).
As Ca2+ influx at the plasma membrane and release from ER are the only two sources for Ca2+ oscillations in HMSCs (Kawano et al., 2002; Kim et al., 2009), we dissected the effect of mechanical force on each process by monitoring the calcium signals at subcellular locations.
Because heat stress has been previously shown to cause mislocalization of FUS, we applied brief periods of thermal stress (35°C for 30 min) and then monitored both the subcellular distribution of FUS and the severity of motor dysfunction.
