PROTAC treatment and immunoblotting

Cells were plated in 6 well dishes (5 × 10⁵–8 × 10⁵ cells) and allowed to attach overnight. Cells were treated with SJF-0628 or SJF-0661 for 20–24 h (unless otherwise stated). The plates were then placed on ice and washed 1x with chilled PBS and lysed in buffer containing 25 mM Tris-HCl [pH 7.4], 0.25% sodium deoxycholate, 150 mM NaCl, 1% Triton X-100, supplemented with protease inhibitors (1x Roche protease inhibitor cocktail) and phosphatase inhibitors (10 mM NaF, 1 mM Na₃OV₄, and 20 mM β-glycerophosphate). Lysates were then cleared at 21,000 g for 10 min at 4 °C. Protein concentrations of the supernatants were then quantified using a Pierce BCA Protein Assay. 12–40 µg of protein were separated using a gradient (4–20%) Criterion TGX precast gel and transferred unto a nitrocellulose membrane. The membranes were then blocked in 5% non-fat milk in TBST (Tris-buffered Saline with Tween 20) for 1 h before probing with the indicated primary antibody overnight. Membranes were imaged using Bio-Rad Image Lab software using ECL prime detection reagent (GE Healthcare, RPN2232 or ThermoScientific, 34095).