Secondary antibodies coupled to horseradish peroxidase

Cells and clinical tissues were lysed for 30–45 mins on ice in lysis buffer containing freshly added protease inhibitor cocktail (Roche Diagnostics, USA). After BCA quantification (Pierce, USA), the protein was added to 5× loading buffer and boiled at 95°C for 10 min. Equal amounts of protein were separated by SDS-PAGE and blotted onto activated polyvinylidene difluoride membranes (Millipore, USA). After blocking, the membranes were incubated with primary antibodies overnight at 4°C. The antibodies used were as follows: BMX (1:2000, 610793, BD), phospho-Etk (Tyr40) (1:500, #3211, Cell Signaling Technology), AKT1 (1:500, sc-5298, Santa Cruz Biotechnology), p-AKT (1:1000, #4060, Cell Signaling Technology), mTOR (1:1000, A2445, ABclonal), p-mTOR (1:1000, AP0094, ABclonal), STAT3 (1:1000, #9132, Cell Signaling Technology), phospho-STAT3 (Tyr705) (1:1000, #4113, Cell Signaling Technology), β-actin and GAPDH (1:1000, Santa Cruz Biotechnology). Blots were incubated with secondary antibodies coupled to horseradish peroxidase (Thermo Fisher Scientific, USA) for 1 h and visualized using ECL detection (Millipore) on X-ray film. Relative quantitation was analyzed with Quantity One software.

The following antibodies were raised by the Division of Signal Transduction Therapy (DSTT) at the University of Dundee in sheep and affinity-purified against the indicated antigens: anti-USP45 (1st, 2nd and 3rd bleed of S052D, residues 756–780 of human USP45) and (1st, 2nd and 3rd bleed of S109D, residues 29–80 of human USP45). Antibody against FLAG M2 clone was from Sigma Aldrich. Total FK2 ubiquitin antibody and K48-linkage-specific ubiquitin antibodies (clone Apu2) was from Millipore and K63-linkage-specific ubiquitin antibodies (D7A11) were from Cell Signaling. Secondary antibodies coupled to horseradish peroxidase were from Thermo Scientific.