Cell lysates were prepared using cold RIPA buffer [150 mM NaCl, 50 mM tris-HCl (pH 8), 0.1% SDS, 1% Igepal, and 0.5% NP-40] containing protease inhibitor cocktail (Roche) phosphatase inhibitors 1 mM Na3VO4 and 10 mM NaF (Sigma-Aldrich) plus 1 mM DTT. Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad). Proteins were separated in 4 to 20% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) (Criterion Precast Gel, Bio-Rad) and blotted onto a nitrocellulose membrane (Amersham, GE Healthcare). Immunoprecipitations were performed using cell lysates in HNTG buffer (20 mM Hepes, 150 mM NaCl, 10% glycerol, and 0.1% Triton X-100) and the indicated primary antibody [anti–green fluorescent protein (GFP) (no. 11814460001, Roche), anti-MYC tag (sc-40 clone 9E10, Santa Cruz Biotechnology), or anti-USP1 (HPA028440, Sigma-Aldrich)] and incubated overnight at 4°C. The immunocomplexes were precipitated by adding protein G or protein A agarose conjugated for an additional 1 hour and 30 min at 4°C. IPs were then washed in HNTG buffer, resuspended in 3× Laemmli Sample Buffer [5× Laemmli buffer composition: 50 mM tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 0.05% bromophenol blue, and 125 mM β-mercaptoethanol], and finally separated on SDS-PAGE for Western blot analysis. Membrane strips were blocked with 5% nonfat dried milk in tris-buffered saline (TBS)–0.1% Tween 20 or in Odyssey Blocking Buffer (LI-COR, Biosciences) and incubated at 4°C overnight with primary antibodies. The following primary antibodies were used: Zeb1 (1:500, #3396 clone D80D3), Slug (1:500, #9585 clone C19G7), pChk1Ser317 (1:500, #12302 clone D12H3), pChk2Thr68 (1:500, #2661), Snail (1:1000, #3879 clone C15D3), pS/TQ (1:1000, #9607 and #2851), p53Ser15 (1:500, #9284), KLF4 (1:500, #4038), c-Myc (1:1000, #5605 clone D84C12), Sox2 (1:1000, #3579 clone D6D9), and β-actin (1:1000, #4970) were from Cell Signaling Technology; Chk1 (1:500, sc-8408 clone G-4), Chk2 (1:500, sc-17747 clone A-11), Twist-1 (1:200, sc-81417 clone Twist2C1a), FANCD2 (1:500, sc-20022 clone FI17), and vinculin (1:5000, sc-7649 clone N-19) were from Santa Cruz Biotechnology; α-tubulin (1:5000, T9026 clone DM1A) and USP1 (1:1000, HPA028440) were from Sigma-Aldrich; and GAPDH (1:1000) and γH2AX (1:500) were from Calbiochem. Antibodies were visualized with appropriate horseradish peroxidase (HRP)–conjugated secondary antibodies (GE Healthcare) for chemiluminescent detection (Clarity, Bio-Rad) or with Alexa-conjugated secondary antibodies (Invitrogen) for Odyssey infrared detection (LI-COR Biosciences).

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