Ab7291

Cell pellets were lysed in 50 mM Tris pH 7.9/8 M Urea/1%Chaps and incubated at 4 °C with agitation for at least 30 min. The lysate was cleared by centrifugation and the supernatant was collected. The protein concentration of the extracts was measured by Bradford assay. About 25 μg of supernatants were resolved by SDS-polyacrylamide gel electrophoresis and transferred onto a Hybond-C Extra Nitrocellulose membrane (Fisher Scientific UK, Loughborough, UK). The membrane was blocked in 5% milk, 0.1% Tween-20 in TBS buffer for 1 h and probed overnight with antibodies against BRG1 (Santa Cruz, sc-17796, dilution 1:2000), a-tubulin (Abcam Ab7291, dilution 1:5000), p21 (Cell Signalling, 2947 S, dilution 1:2000), INO80 (Bethyl, A303-371A, dilution 1:2000) in 5% milk, 0.1% Tween-20 in TBS buffer. The membrane was washed three times with 0.1% Tween-20 in TBS and incubated for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibodies in 5% milk, 0.1% Tween-20 in TBS. The secondary antibodies used were Rabbit anti-Mouse HRP (Dako, P0260, dilution 1:5000) and Goat anti-Rabbit HRP (Dako, P0448, dilution 1:5000). Proteins were visualised by using in-house ECL reagent or SuperSignal West Pico Chemiluminescent Substrate (Life Technologies).

Oocytes were lysed in SDS sample buffer at 95°C for 8 min and subjected to SDS-PAGE. Samples were transferred to PVDF membranes, and blocked in TBST (TBS containing 0.5% Tween 20) with 1% BSA for 1 h at room temperature. Membranes were incubated with primary antibodies overnight at 4°C. After three washes in TBST, membranes were incubated with secondary antibodies for 2 h at room temperature. The blots were developed with the ECL Plus Western blotting detection kit (GE Healthcare). Primary antibodies for immunoblotting were anti-RASSF1A antibody (Abcam, ab23950, 1:500), anti-acetylated-α-tubulin (acetyl K40) antibody (Sigma, T7451, 1:500), anti-α-tubulin antibody (Abcam, ab7291, 1:500), anti-HDAC6 antibody (Cell Signaling, #7612, 1:500), anti-SIRT2 antibody (Abcam, ab67299, 1:500) and anti-β-actin antibody (Cell Signaling, #4967, 1:2,500). Secondary antibodies were HRP-conjugated anti-rabbit or anti-mouse antibodies (Jackson ImmunoResearch, 111-035-144, 111-585-144, 1:2,500).

Cultured cells were treated with lysis buffer on ice, centrifuged at 15,000g, and supernatant collected. Protein content was quantified using the bicinchoninic acid assay. Lysates were loaded on 7.5% acrylamide gels, and subjected to SDS-PAGE to separate proteins. Proteins were transferred to PVDF membrane, incubated with blocking buffer (LI-COR, Cambridge, UK) for 1h, and incubated overnight with primary antibody diluted in blocking buffer. Primary antibodies targeting RFWD3 (Abcam, Cambridge, UK; ab138030; 1:500 dilution), tubulin (Abcam; ab7291; 1:5000 dilution), PARP (Cell Signaling Technology, Danvers, MA, USA; 9542; 1:1000 dilution) and FANCD2 (Abcam; ab108928; 1:1000 dilution) were used. Membranes were incubated with anti-mouse IRDye 680LT (LI-COR 926-68020) and anti-rabbit IRDye 800CW (LI-COR 926-68023), and imaged using the Odyssey Infrared Imaging System (LI-COR Biosciences). Quantification of band intensity was performed using ImageJ v1.53 (24 (link)).