Horseradish peroxidase linked anti mouse secondary antibody

OS cells were collected, washed with 1X Dulbecco's phosphate‐buffered saline (Gibco) and re‐suspended in complete lysis buffer consisting of 20 mM Tris‐HCL pH 8.0, 137 nM NaCl, 10% glycerol, 1% IPEGAL CA‐630, 10 mM ethylenediaminetetraacetic acid, 1 mg/mL aprotinin, 1 mg/mL leupeptin, 1 mg/mL pepstatin A, 1 mM phenylmethylsulphonyl fluoride, 1 mM sodium orthovanadate, and 10 mM sodium fluoride for 1 hour at 4°C for protein extraction. Frozen tumours were pulverized and re‐suspended in complete lysis buffer for 1 hour at 4°C. Extracted protein was quantified using the Bradford assay and 75 μg protein was separated by SDS‐PAGE and transferred to PVDF membranes. Membranes were blocked in TBST containing 5% non‐fat dry milk for 1 hour at room temperature and incubated overnight with anti‐XPO1 (5G3, Invitrogen, Cat.#MA5‐27879, 1:40 000 dilution) at 4°C. Membranes were incubated in horseradish peroxidase linked anti‐mouse secondary antibody (Cell Signalling, Cat.#7076), washed, and exposed to substrate (SuperSignal West Dura Extended Duration Substrate, Pierce, Rockford, Illinois). Blots were stripped (Restore, Pierce), washed, and re‐probed for β‐actin (8H10D10, Cell Signalling, Cat.#3700, 1:20000 dilution) overnight at 4°C. Band intensities were calculated with ImageJ software (NIH, Bethesda, Maryland) and relative intensity of XPO1 was determined by dividing by β‐actin.