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We next asked whether the MRN complex contributed to the formation of the slower migrating forms of OriP DNA.
The effects caused by Nhtt138Q appeared to be more prominent, especially the slower migrating forms of the protein (indicated as gp78* in Fig. 1A).
We refer to the most slowly migrating forms of Shs1 as the upper forms, and the most rapidly migrating form of Shs1 as the lower form (Figure 4B).
Nonetheless, these mutants retained the ability to interact with poly-ubiquitylated HIF1α (that is, the slowest migrating forms of HIF1α), presumably via the UBA domain.
This technique allowed detectable binding of only the fastest SDS-PAGE migrating forms of 4E-BP1, indifferentialferential binding between hypophosphorylated and hyperphosphorylated 4E-BP1 had occurred.
Surprisingly, immunoblotting of total RIP1 revealed slower migrating forms of RIP1 at 3 h after TBZ but not after the apoptotic stimulus of TNF α and BV6 or the individual stimuli.
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We noted the appearance of faster migrating forms on this blot.
Moreover, we observed retention of the slower migrating form of H-Ras on RBD beads upon treatment with PAO.
A reduction in the levels of the slower migrating form of H-Ras was also observed with high (10 µM) PAO concentrations (results not shown).
Third, western blotting indicates a faster migrating form of Gln3p in these cells, consistent with reduced phosphorylation of this physiological substrate of TORC1.
Interestingly, when the S365A mutation was introduced into Osw3-HA and this protein was examined, only the slower migrating form of the protein was seen (Figure 8C).
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