- Grow HeLa, HEK293, and RAW264.7 cells in DMEM (Thermo Fisher Scientific, 41965-039, lot 1924339) supplemented with 10% FBS (Merck Millipore, S0115, lot 1248D) and antibiotics (1x Penicillin-Streptomycin, Thermo Fisher Scientific, 15140-122, lot 1970744), at 37°C and 7% CO2 in 6-well plates. Grow PK-15 cells in DMEM supplemented 5% FBS and antibiotics.
- Transfect 5x105 HeLa cells/well with plasmids expressing either HA-FUBI, HA-FAU (FUBI+S30), or empty pcDNA3.1 vector using X-Treme Gene HP transfection reagent (Roche, 06366236001, lot 28049700; 3µg plasmid/well, ratio DNA to X-Treme Gene HP 1:1) following the manufacturer´s instructions. Optional: treat cells with 2.5µM proteasome inhibitor MG132 (Biozol, CAY-10012628) for 16h before lysis.
- Lyse cells in ice-cold lysis buffer containing 20mM HEPES pH 7.4, 1% Triton X-100 (v/v), 5% glycerol (v/v), 8mM EDTA, 2mM EGTA, Complete protease inhibitor cocktail (Roche, 11873580001, lot 31880700).
- Centrifuge lysates at 16000rcf for 10min at 4°C to pellet cell debris and collect supernatant.
- Determine protein concentration using Bradford reagent (Thermo Fisher Scientific, 1856210, lot MH160588) and BSA as a standard.
- Denature proteins for 5min at 95°C in 1x Laemmli SDS sample buffer.
- Separate 20µg of total protein per lane on a 16% Tris-glycine mini gels (Bio-Rad, freshly cast 8.3cm x 7.3cm for Mini Protean Tetra Cell) using Tris-glycine running buffer (Tris: Fisher Scientific, BP152-5, lot 171324; Glycine: AppliChem, A1067, lot 7A015185) in a Mini Protean Tetra Cell for 75min at 160V.
- Transfer proteins onto nitrocellulose membrane (LI-COR, 926-31092, lot 25452956) by semi-dry blotting (Bio-Rad Trans-Blot SD) using discontinuous Tris-CAPS buffer system (Bio-Rad; CAPS: Sigma-Aldrich, C2632, lot SLBR8754).
- Block membrane either with Rotiblock (Carl Roth, A151.4, lot 297261307), 5% milk in PBS, or 1% BSA in PBS for 45min at RT.
- Incubation with primary
- rabbit anti-FAU antibody (antibodies-online, ABIN2798885, lot RAY8070723) diluted 1:1000 in PBS/0.1% Tween 20 ON at 4°C or
- rabbit ant-HA antibody (Abcam, ab9110, lot GR235874-6) diluted 1:5000 in PBS/0.1% Tween 20 ON at 4°C.
- Wash membrane 3x for 10min with PBS/0.1% Tween 20.
- Incubate membrane with secondary IRDye88CW goat anti-rabbit IgG (H+L) antibody (LI-COR, 926-32211) diluted 1:10000 in PBS/0.1% Tween 20 for 1h at RT.
- Wash membrane 3x for 10min with PBS.
- Acquire image on a LI-COR Odyssey CLX scanner.