To verify whether the Ozz-MyHCemb interaction occurred in vivo, crude lysates of proliferating (day 0), differentiating (day 2) and terminally differentiated (day 4) primary myoblast cultures prepared from newborn wild-type mice were immunoprecipitated with anti-MyHCemb antibody or an isotype matching control IgG, and probed on immunoblots with anti-Ozz antibody.
Interestingly, cell fractionation showed no increase in nuclear localization of β-catenin in undifferentiated (day 0) vs. differentiated (day 2) cultures (Figure S3), suggesting regulation distal to nuclear translocation.
To test whether IGF-2 was able to stimulate mitogenesis of brown adipocytes, we exposed postconfluent (day 2) and differentiated (day 7) T37i cells to 20 nM IGF-2 or insulin (which served as a positive control) and measured 3H-thymidine incorporation (Fig. 5A).
In contrast, PC12 cells on nanostructured microprobes were not surface-bound on the 1-day in vitro culture (DIV1, 1 day after seeding), but differentiated 3 days after NGF treatment (Fig. 5b, c).
Total RNAs from undifferentiated (Day 0), neuronally differentiated P19 (Day 7), and early glial stage cells (Day 10) were applied to a GeneChip Mouse Exon 1.0 ST Array (Affimetrix).
A GeneChip Exon Array was performed using total RNAs purified from undifferentiated P19 cells (Day 0), neuronal differentiated cells (Day 7), and cells from the early glial stage (Day 10).
Consistent with previous reports [31], [32] hPSC colonies differentiated 7 days following Oct4 depletion (Figure S1I L).
SH-SY5Y cells were differentiated 6 days with 10 µM retinoic acid [39] and subsequently fixed with 4% paraformaldehyde in PBS.
Its increase as well as that of RXRA is also detectable in long-term differentiated (30 days) primary human adipocytes [23].
All cells died or differentiated within days.
Total RNA was isolated from differentiated (7 days) wild-type/P1-deficient myotubes using TRIzol reagent (Invitrogen) according to the manufacturer's instructions.
