Mouse anti p62

The following antibodies were used for detection of proteins in GFP-TRAP experiments on HeLa lysates are. Mouse anti-p62 (BD Bioscences, #610832, Franklin Lakes, NJ, USA) was used at 1:1000 dilution; Rabbit anti-NDP52 (Cell Signaling, #9036) was used at 1:1000 dilution; Rabbit anti-OPTN (Sigma, HPA003279) was used at 1:500 dilution; Mouse anti-GFP (Roche, cat. 11 814 460 001) was used at 1:1000 dilution; Mouse anti-GAPDH (Sigma, Clone GAPDH-71.1) was used at 1:25000 dilution. HRP-conjugated goat anti-Rabbit and anti-Mouse (Jackson
ImmunoResearch, #111-035-003 and 115-035-003, respectively) were used at 1:10000 dilution.

HeLa cells were seeded in 10 cm dishes and let grow until 80% confluency. Cells were transfected with pME18s vectors (Addgene) containing HA-FIP200 (wt, point mutants or truncation) using Fugene transfection reagent (Promega). A vector:Fugene ratio of 1:6 was used for transfection. 24 h after transfection, cells were harvested with trypsin. The cell pellets were washed in PBS and resuspended in 100 μl lysis buffer (20 mM HEPES pH 7.5, 250 mM Sorbitol, 0.5 mM EGTA, 5 mM Mg-Acetate, 0.3 mM DTT, cOmplete EDTA-free protease inhibitor cocktail (Roche)). After 1 freeze and thaw cycle, the lysates were clarified by spinning at 1,000xg for 10 min at 4°C. Protein concentration in the lysates was measured by Bradford assay (BioRad) and all samples were adjusted to the same final concentration in 300 μl IP buffer (25 mM HEPES pH 7.5, 125 mM NaCl, 0.05% Triton X-100). Anti-HA magnetic beads (Pierce – Thermo Scientific) were washed 3 times in IP buffer and 1,5 μl of beads slurry were incubated with each sample for 1 h at 4°C on rotating wheel (9 rpm). After 3 × 5 min washes in IP buffer, beads were resuspended in 10 μl of 2x non-reducing protein loading dye and heated for 10 min at 95°C.
Samples were analyzed by western blot with mouse anti-p62 (1:500 – BD Bioscience) and rabbit anti-FIP200 (1:1000 – Atlas antibodies).