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Example sentences for: ha-pp
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The doxycycline-independent increase in 6His-HA-PP1α is attributed to background expression when using this system [ 25 ] . As predicted for a protein under the control of the doxycycline-inducible promoter, the abundance of 6His-HA-PP1α decreases in the absence of the inducer over time (Figure 2C).
As expected [ 25 ] , some low-level expression of 6His-HA-PP1α in the absence of the inducer doxycycline can also be detected.
To assay pRB-directed phosphatase activity, [ 32P]-labeled pRB substrate was prepared by immunoprecipitation with monoclonal antibody to pRB, PMG3-245 (PharMingen), from [ 32P]-labeled CV-1P cells as described previously [ 20 ] . For the source of enzyme, immunocomplexed 6His-HA-PP1α from induced LLWO2F cells were mixed and incubated with [ 32P]-labeled immunoprecipitated pRB for 30 minutes at 30°C as previously described [ 20 ] . Phosphatase reactions were terminated by the addition of SDS-PAGE sample buffer and boiling.
Immunoprecipitation using PP1α antibody followed by western blotting using the same anti-PP1α reveals a reciprocal relationship in PP1α abundance following doxycycline induction; endogenous PP1α levels decrease in response to increasing levels of 6His-HA-PP1α (top panel, compare left two lanes).
Parallel experiments performed using the same lysate and antibody to hemagglutinin for immunoprecipitation and western blotting show the position of 6His-HA-PP1α and the fact that endogenous PP1α is not recognized by this antibody (bottom panel).
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