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The aberrantly migrating samples were re-amplified using sequencing primers and sequencing was performed using Big Dye Cycle-sequencing kit according to the manufacturer's instructions.
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Two positive samples were found, one from a migrating Tennessee warbler and another from an American robin.
To test whether this slower migrating band represented a glycosylated form of Fam20a, the protein samples were treated with the enzyme N-glycosidase F (PNGaseF).
Samples were probed with anti-Pex5p antibodies and slower migrating Pex5p species appeared.
For analysis by LC-MS/MS, protein samples were briefly run into the resolving gel to separate out the faster-migrating FLAG peptide.
Such mobility shift indicated that the fast migrating band in vernalized samples is the dephosphorylation form of VER2.
Both in SH-SY5Y cells (Fig. 5B) and in htau neurons (Fig. 5C), a protein of ∼50 kDa, co-migrating with tau from whole cell lysates (input samples), was detected in cells transduced with LV-TauPTM6 at m.o.i.o.i
To test if the slower migrating species of Yox1p-HA present in HU-treated samples are the result of phosphorylation, immunoprecipitated Yox1-HA was treated with λ-phosphatase.
Interestingly, when a Pi-affinity PAGE immunoblot was probed with anti-pSer, only the slowest migrating band in the untreated co-immunopurified sample was immunoreactive.
The slower-migrating yellow green-fluorescing spot, but not the blue spot, was partiallyellow green-fluorescinglactonized spotle was treated at pH <1, which promotes re-lactonization.
Higher migrating bands were detected and the intensity of these bands was greater in the sample from cells expressing the dominant negative Hsc70 mutant (Figure 10C).
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