Nitrocelulose membrane

Cell lysates were mixed 1:1 with 2 × SDS-PAGE sample buffer and incubated for 5 min at 95 °C. Afterwards, they were separated by SDS-PAGE electrophoresis and subsequently electrotransferred onto nitrocelulose membrane (Amersham). Membranes were blocked with 5% BSA (Bioshop) in Tris-Buffered Saline with Tween 20 (TBST). Membranes were incubated with primary antibodies (Caveolin-1 N-20 Antibody (sc-894, Santa Cruz Biotechnology) diluted 1:1000 in 2.5% BSA in TBST and β-tubulin Antibody (sc-134234, Santa Cruz Biotechnology) diluted 1:2000 in 2.5% BSA in TBST for 2 h and washed in TBST. Subsequently, membrane was incubated with HRP-labeled anti-rabbit IgG antibody (A0545, Sigma) diluted 1:20 000 in 1% BSA in TBST) for 1 h and finally the signal was developed using the ECL system (Amersham).

Protein was extracted using the AllPrep DNA/RNA mini kit (Qiagen) and resuspended in 5% SDS. The protein concentration of the supernatants was determined using BCA kit (Pierce). Total lysates of 14 μg of protein were denatured in NuPage LDS sample buffer and NuPage reducing reagent by heating for 10 min at 95 °C. Proteins were separated on NuPage 4–12% Bis–Tris gels using MOPS running buffer (Thermofisher). Wet transfer onto a nitrocelulose membrane (Amersham Biosciences) was performed using MOPS running buffer with 20% methanol. Membranes were blocked with 10% milk/1% BSA in Tris-buffered saline (TBS)/0.1%Tween (TBS-T) overnight at 4 °C. Primary antibodies mouse anti-TET3 (1:1000, Abcam, ab174862) and mouse anti-α-Tubulin (1:5000, Sigma-Aldrich, T6074) diluted in blocking buffer and incubated 2 h at RT. Membranes were washed in TBS/T and incubated with the secondary antibody coupled to horseradish peroxidase (BioRad) 1 h at RT. The bound antibodies were visualized by chemiluminescence using ImageQuant LAS4000 mini (GE Healthcare). Bands were analysed using ChemiDoc (Bio-Rad) and quantification was performed with ImageLab software (Bio-Rad). α-Tubulin was used as loading control.