Bolt sample reducing agent buffer

Cells were homogenized in RIPA buffer with protease and phosphatase inhibitor cocktails (Roche, 11836153001, 04906837001). Fractions that were soluble and insoluble in 1% Triton X-100 were obtained by centrifugation at 100,000 × g for 30 min at 4°C. Supernatants containing the soluble fractions were harvested, and the pellets for insoluble fractions were solubilized in 2% SDS detergent Cell Lysis Buffer (Cell Signaling Technology, 9803). After sonication, the cell lysates were mixed with 4× Bolt LDS Sample buffer (Invitrogen, B0007) and 10× Bolt Sample Reducing Agent buffer (Invitrogen, B0009), and then they were boiled at 95°C for 5 min.

Cells were homogenized in Cell Lysis Buffer (Cell Signaling Technology, 9803) containing protease and phosphatase inhibitor cocktails. Protein concentrations of the cell lysates were determined by BCA protein assay (Thermo Fisher Scientific, 23225). Next, the protein extracts were mixed with 4× Bolt LDS sample buffer (Invitrogen) and 10× Bolt Sample Reducing Agent buffer (Invitrogen), and then they were boiled at 95°C for 5 min. An equal amount of protein from each sample was separated on Bolt 4–12% Bis-Tris gels (Invitrogen, NW04120BOX) or NuPAGE 3-8% Tris-Acetate gels (Invitrogen, EA0378BOX), and then it was transferred to polyvinylidene difluoride (PVDF, Invitrogen, LC2005) membranes. After blocking membranes with 5% skim milk in TBS with 0.025% Tween 20, blots were probed with antibodies as indicated and detected with an ECL prime kit (GE healthcare, RPN2232). Samples from three independent experiments were used, and the relative expression levels were determined using a Fusion-FX imaging system (Viber Lourmat).