The rabbit polyclonal antibodies specific for USP1 phospho–serine 42 (pS42) and phospho–serine 331 (pS331) were generated by GenScript Inc. Two New Zealand rabbits for each phospho-antibody were immunized with specific keyhole limpet hemocyanin (KHL)–conjugate peptides. The sequences of KHL-conjugate peptides used for rabbit immunization were as follows: USP-1-S42, CTKRALDFTDSQENE; USP-1-pS42, CTKRALDFTD{pSER}QENE; USP-1-S331, CSENESPRPSQKKSR; and USP-1-pS331, CSENESPRP{pSER}QKKSR.

Validation of the phospho-antibodies’ specificity was performed by enzyme-linked immunosorbent assay (ELISA) and approved by the Antibody Department of GenScript Inc. We tested the specificity of the two USP1 phosphorylation-specific antibodies in Western blot analysis as follows. Lysates (400 μg for each sample) from OVCAR-8 USP1 KO cells or USP1 KO cells stably expressing USP1WT or USP1S42A/S331A mutant were immunoprecipitated with an anti-USP1 antibody (HPA028440, Sigma-Aldrich) as described in the “Preparation of cell lysates, immunoblotting, and immunoprecipitation” section. Membranes were incubated with pS42- or pS331-USP1 (1:500) primary antibodies diluted in 5% (w/v) bovine serum albumin (BSA), 1× TBS, and 0.1% Tween 20, at 4°C overnight. Membranes were then probed with anti-rabbit HRP-conjugated secondary antibodies (1:2000) (GE Healthcare) and processed by chemiluminescence.

For detection of endogenous phosphorylation of S42-USP1 or S331-USP1, lysates (600 μg) from MDAH-2774 and OVCAR-8 cells, treated or not with CDDP for the indicated times, were immunoprecipitated as described above, and membranes were incubated with pS42-USP1 (1:500) or pS331-USP1 primary antibodies diluted (1:250) in 5% (w/v) BSA, 1× TBS, and 0.1% Tween 20, at 4°C overnight.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.