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Figure 3.
Figure 3. Mass Spectrometry Confirms the Linkage Specificity
of Apu2.07 and Apu3.A8 in Immunoprecipitations
(A–D) BJAB
cell lysates were immunoprecipitated with Apu2.07, Apu3.A8, or
an isotype control antibody recognizing HER2. Mass spectrometry
was used to determine the total amount of ubiquitin
immunoprecipitated (A) as well as the polyubiquitin linkages in
the lysate (B) and immunoprecipitates (C and D).
(E) MuRF1
autoubiquitination reactions performed in vitro with WT, K48R,
or K63R ubiquitin were immunoprecipitated with Apu2.07, Apu3.A8,
or isotype control. Numbers in parentheses indicate the relevant
lanes and columns in (F)–(I).
(F) Autoubiquitination
reactions and immunoprecipitations depicted in (E) were western
blotted with a pan-ubiquitin antibody. The hatched red lines
indicate the portion of the gel that was cut out and subjected
to analysis by mass spectrometry.
(G–I) Mass spectrometry
was used to determine the polyubiquitin linkages in the
autoubiquitination reactions and immunoprecipitations depicted
in (E).
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