Whole HeLa cell extracts were prepared using 1X Laemmli SDS sample buffer. The cells were washed twice in 1X PBS and incubated in 1X Laemmli SDS sample buffer for 30 min on ice. Subsequently, the samples were boiled at 96°C for 15 min and subjected to SDS-PAGE using bis-tris 4–12% gradient gels (Thermo Fischer NP0301BOX; NP0302BOX; NP0303BOX) or tris-acetate 3–8% gradient gels (Thermo Fischer EA0375BOX; EA03752BOX; EA03755BOX).
The proteins were subsequently transferred from the gel to a PVDF membrane (Millipore IPFL00010) for immunoblotting. The membranes were blocked in 5% non-fat milk powder resuspended in TBS supplemented with 0.1% Tween 20 (TBS-T) for 1 h at RT or overnight at 4 C, followed by incubation with antibodies. The membranes were developed with Luminata Forte (Millipore WBLUF0500).
The proteins were subsequently transferred from the gel to a PVDF membrane (Millipore IPFL00010) for immunoblotting. The membranes were blocked in 5% non-fat milk powder resuspended in TBS supplemented with 0.1% Tween 20 (TBS-T) for 1 h at RT or overnight at 4 C, followed by incubation with antibodies. The membranes were developed with Luminata Forte (Millipore WBLUF0500).