*2.7. Western Blotting*

Cells were lysed in RIPA buffer (Sigma-Aldrich, R0278, Saint Louis, MO, USA) containing 1 tablet of protease inhibitor (Sigma-Aldrich, 11836153001, Saint Louis, MO, USA) and 1 tablet of phosStop (Sigma-Aldrich, 04906837001, Saint Louis, MO, USA) per 10 mL. The total protein concentration in the lysates was determined using a Pierce BCA Protein Assay kit (Thermo Scientific, 23225, Waltham, MA, USA). The absorbance was measured using a microplate spectrophotometer (Epoch Biotek, Winooski, VT, USA) and the protein concentration was calculated using Gen5 software (BioTek, Winooski, VT, USA). An 8% polyacrylamide gel was made. Then, 15 μL sample mix, containing 9 μg total protein and 5 μL sample buffer (5.7 mL water, 1.6 mL glycerol, 1.1 mL 10% SDS, 1.3 mL 0.5 M Tris (pH6.8), 25 mg dithiotreitol (DTT), 300 μL bromophenol blue) was heated to 90 ◦C for 5 min, cooled on ice and loaded onto the gel. The gel was run in electrophoresis buffer (25 mM Tris base, 190 mM glycine, 0.1% SDS) at 70 V until the samples reached the separating gel and then run at 150 V until the samples reached the bottom of the gel. Next, the proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (GE Healthcare, 15269894, Chicago, IL, USA) for 1 h at 80V on ice in cold blot buffer (25 mM Tris base, 190 mM glycine, 20% ethanol). The membrane was rinsed in water and washed 2x in Tris-buffered saline and Tween-20 (TBS-T) (20 mM Tris/HCl, 137 mM NaCl, 0.1% Tween-20). The membrane was incubated for 1 h in 2% enhanced chemiluminescence (ECL) prime blocking agent (GE Healthcare, RPN418, Chicago, IL, USA) in TBS-T at 4 ◦C while shaking. Subsequently, the membrane was incubated overnight in 2% blocking agent in TBS-T with primary antibody (Table 2) at 4 ◦C while shaking. The membrane was washed 3 × 5 min in TBS-T and incubated in 2% blocking agent in TBS-T with secondary antibody (Table 2) for 1 h at room temperature. ECL solution A and B (GE Healthcare, RPN2235, Chicago, IL, USA) were mixed 1:1 at room temperature and the membrane was incubated for 5 min. Images were taken by the ImageQuant LAS500 (GE healthcare, life sciences, Chicago, IL, USA) and relative intensity of protein bands was quantified using ImageJ [26]. Pan actin was used as a loading control.



#### *2.8. Immunofluorescence*

Cells were washed 2x with cold phosphate-buffered saline (PBS) (Gibco, 14190250, Waltham, MA, USA) and fixated for 10 min in 4% paraformaldehyde (PFA) (Fisher Scientific, Pittsburgh, PA, USA) at room temperature. Cells were washed 3x in PBS and permeabilised in 0.1% Triton X-100 in PBS for 10 min. After this, the cells were washed 3x in PBS with 0.05% Tween20 (PBS-T) and incubated for 1 h in 5% normal goat serum (ThermoFisher Scientific, 50062Z, Waltham, MA, USA) in PBS at room temperature. The cells were incubated overnight with primary antibody (Table 2) in 5% normal goat serum in PBS at 4 ◦C. Then, the cells were washed 3 × 5 min in PBS-T and incubated with secondary antibody (Table 2) in PBS-T for 1 h at room temperature. Cells were washed again 3 × 5 min in PBS-T and incubated in PBS with 4',6-diamidino-2-phenylindole (DAPI) (100 ng/mL). After this, the cells were rinsed with PBS and stored in PBS at 4 ◦C. Images were taken with a fluorescent microscope (Zeiss Axiovert 200M, Hyland Scientific, Stanwood, WA, USA) using the program Slidebook 5.0 (Intelligent Imaging Innovations, Göttingen, Germany). The images were analysed using ImageJ [26].
