Ab4074

Protein extracts of mouse ventricles or fibroblasts were prepared in RIPA buffer containing protease-inhibitor and phosphatase-inhibitor tablets (Roche, Vienna, Austria). After mechanical disruption of tissue samples, equivalent amounts of protein were separated on a SDS polyacrylamide gel, followed by electro-transfer to nitrocellulose membrane. Nonspecific antibody binding was blocked by incubation in 5% (m/v) non-fat dry milk powder in TBS-T (20 mM Tris-Cl, pH 7.5, 150 mMNaCl, 0.1% (v/v) Tween 20) at room temperature for 1 h. Then, samples were incubated overnight at 4°C with antibodies: anti-ABCG2 (#ab3380, Abcam) and anti-α-tubulin (#ab4074, Abcam) for ventricular tissues, and anti-ABCG2 (#AV43649, Sigma-Aldrich, Saint Louis, USA) and anti-GAPDH (#sc-25578, Santa Cruz Biotechnology, Heidelberg, Germany) for fibroblasts. After 1 h incubation in room temperature with peroxidase-labeled secondary antibody (Pierce Biotechnology, Rockford, USA), specific immunoreactive signals were detected using Enhanced ChemiLuminescence kit (Amersham Biosciences, Buckinghamshire, UK) or West Pico Substrate (Thermo Scientific, Rockford, USA).

Whole‐cell extracts were prepared in radioimmune precipitation assay (RIPA) buffer (50 mmol/L Tris‐HCl [pH 7.5], 150 mmol/L NaCl, 1% NP‐40, 0.1% SDS, 0.5% sodium deoxycholate, 0.5 mmol/L DTT, 0.5 mmol/L PMSF and 2.5 mmol/L Roche protease inhibitor cocktail). The extracts were subjected to Western blot analysis as described previously.¹¹ Briefly, 20 to 40 μg of total protein were separated using 7‐12% SDS‐PAGE and transferred to PVDF membrane. The membrane containing transferred proteins was blocked by incubating with 5% BSA containing 0.05% Tween‐20 and incubated overnight at 4°C with primary antibody to detect CYCLIN D1 (Abcam, ab134175), c‐MYC (Abcam, ab62928), SURVIVIN (Abcam, ab76424), α‐TUBULIN (Abcam, ab4074), DKK1 (Abcam, ab109416), DKK3 (Abcam, ab186409), β‐ACTIN (Cell Signaling Technology, 4970), CYCLIN D3 (Cell Signaling Technology, DCS22) and PRMT5 (Thermo Fisher, MA1‐25470). After incubation with primary antibody, the membrane was treated with HRP‐conjugated goat anti‐mouse (Amersham Biosciences, NA931) or anti‐rabbit (Amersham Biosciences, NA934V) secondary antibody. Next, proteins were visualized using the ECL detection kit (Amersham, RPN2209) in a Western blot imager (Flurochem E system, proteinsimple).

Proteins were detected via SDS-PAGE and Western blotting as described in Horos et al.^{4 (link)}. Antibodies used were directed against Csde1 (NBP1-71915, Novus Biological), Actin (A3853, Sigma-Aldrich) and alpha Tubulin (ab4074, Abcam). Fluorescently labeled secondary antibodies for visualization with Odyssey were IRDye 680RD Donkey anti-Rabbit IgG (926–68073, Licor) and IRDye 800CW Donkey anti-Mouse IgG (925–32212, Licor).

Primary antibodies used included those against α-tubulin (T5168, Sigma-Aldrich, ascites fluid, 1:5000; ab4074, Abcam, 1 μg ml⁻¹), β-tubulin (ab21057, Abcam, 1 µg ml⁻¹), spastin (ab31850, Abcam, 10 µg ml⁻¹; Fig. S1A-C), polyglutamylated tubulin B3 (T9822, Sigma-Aldrich, 2 µg ml⁻¹), γH2AX (05-636, Millipore, 0.1 µg ml⁻¹), cleaved caspase 3 (9664, Cell Signaling Technology, 0.5 μg ml⁻¹) and cleaved-caspase 9 (9509, Cell Signaling Technology, 1 μg ml⁻¹). Secondary antibodies included Alexa Fluor 488 donkey anti-goat (A11055, Invitrogen), Alexa Fluor 555 donkey anti-goat (A21432, Invitrogen), Alexa Fluor 555 donkey anti-mouse (A31570, Invitrogen), Alexa Fluor 647 donkey anti-mouse (A31571, Invitrogen) and Alexa Fluor 647 donkey anti-rabbit (A31573, Invitrogen). Parallel sections were processed in the absence of a primary antibody to control for secondary antibody specificity.