All samples were collected and processed within four hours of expression.
To confirm this observation, we used a time course experiment to monitor CFU formation during eight hours of expression and eight hours of recovery.
After four hours of expression, appropriate dilutions of cells were plated to YPD agar to determine viability after HO cutting, and SC-His to select for recombinants.
In transduced myoblasts significant conversion to the LC3b-II form was observed after 16 24 hours of expression of YFP-hDMPK A, but not C (Figure 4D).
After 24 hours of expression, cells were harvested by centrifugation at 5000 g for 20 minutes, and stored at −20°C for future use.
After four hours of expression, the cells were plated to medium lacking histidine to select for recombinants, and dilutions plated to YPD to determine viability.
To confirm the plasma membrane insertion of STIM1 in neurons we expressed STIM1-GFP in cultured dissociated hippocampal neurons by Sindbis virus transduction, and after 12 hours of expression, the cells were live-labeled with anti-GFP antibodies.
As can be seen in Fig. 1A (right bottom panels), within 48 hours of expression of the mutant protein, a fraction of HeLa cells also showed enlarged and flattened morphology, which was not observed in cells transfected with the vector control (Fig. 1A, bottom left panel).
For each plasmid, 100 µl was taken from the cultures after 24 hours of expression, diluted in five ten-fold steps from each of which 100 µl was spread on LB-agar plates and LB-agar-ampicillin plates for overnight culture at 37°C.
Finally, an intriguing fly-to-fly variability in reporter expression patterns was observed: the hsp70 and hsp22 transgenic reporters consistently exhibited ~24 hour or ~18 hour periods of expression, respectively.
