Ab176727

Cells were re-suspended in the lysis buffer (50 nM Tris-HCl pH = 7.6–8.0, 0.5% NP40, 1 nM ethylene diamine tetraacetic acid, and 1 mM b-mercaptoethanol) containing a protease inhibitor, and then subjected to ultrasonic lysing. Following 10-min centrifugation at 14000 g, the supernatant was incubated overnight with antibody anti-PTEN (ab267787, Abcam) or anti-IgG (ab172730, Abcam) and protein A/G agarose beads at 4°C overnight. After washing with the lysis buffer thrice, the beads were boiled in 40 μL 2 × SDS-PAGE sample buffer and then subjected to SDS-PAGE, followed by Western blot assay with the anti-OTUD5 (ab176727, Abcam) and HRP-coupled secondary antibody (ab6721, Abcam). Afterward, the protein bands were visualized using the chemiluminescence method.

Total protein content was extracted from cells using a radioimmunoprecipitation assay lysis buffer (Invitrogen) and quantified with the bicinchoninic acid method. Subsequently, 50 μg proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to nitrocellulose membranes. The membranes were subsequently blockaded with 5% non-fat milk and incubated with antibodies anti-OTUD5 (dilution ratio of 1:2000, ab176727, Abcam, Cambridge, MA, USA), anti-PTEN (dilution ratio of 1:1000, ab267787, Abcam), and anti-GAPDH (dilution ratio of 1:1000, ab9485, Abcam) at 4°C overnight. Following rinsing with Tris Buffered Saline Tween (TBST) thrice (15 min each time), the membranes were cultured with horseradish peroxidase (HRP)-coupled secondary antibody (dilution ratio of 1:2000, ab6721, Abcam) at room temperature for 1 h. Afterward, the membranes were washed with TBST thrice (15 min each time), reacted with an enhanced electrochemical luminescence reagent, and imaged. The gray value was analyzed using the Image J software, with GAPDH serving as the internal reference.