Immunoprecipitation of phosphorylated proteins

HEK293 were plated at a density 5x10⁵ cells per well of a 6 well plate (2 wells were used per condition) and transfected the next day with K15-encoding or empty PFJ vector using Fugene. HEK293 BAC36 WT/ΔK15 cells were plated at density 3x10⁶ per T75 flask and HuART BAC36 at density 5.8x10⁶ cells per 15 cm culture dish. After 24 hours virus-infected cells were reactivated and 48 hours after reactivation washed with PBS and lysed in 600 μl (for HEK293) or 500 μl (for HuART) Tris buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% (v/v) NP-40, protease and phosphatase inhibitors). For the transfected cells 300 μl Tris buffer was used per well of a 6-well plate (48 hours after transfection). Lysates were incubated on ice for 30 minutes and cleared by centrifugation for 10 minutes at 20.000g, 4°C. Phosphotyrosine affinity beads (30 μl per IP) (Cytoskeleton, Inc.) were washed twice in 1 ml of PBST and centrifuged at 800g, 4°C for 1 minute. The lysates (1.8 mg per IP) were incubated overnight at 4°C, washed 3 times with 1 ml Tris buffer and the bound proteins were eluted for 5 minutes at room temperature in 30 μl 2x non-reducing SDS sample buffer (125 mM Tris pH6.8, 20% (v/v) glycerol, 4% (v/v) SDS, 0.005% Bromphenol Blue). The samples were centrifuged at 20.000g for 1 minute, supernatants were incubated for 5 minutes at room temperature with 1μl β-mercaptoethanol and analysed by western blot.

For co-immunoprecipitation, 1 μg of pFJ K15P and 1 μg of pTriex-4 PLCγ1 SA were co-expressed in HEK-293T cells. Thirty-two hours after transfection cells were washed once with PBS and lysed in 300 μl IP buffer per well of a 6-well plate. Lysates (500 μl) were incubated overnight at 4°C with 10μl Red Anti-Flag M2 beads (Sigma Aldrich), which were washed before 3 times with 300 μl IP buffer. After that, beads were subjected to 6 washing steps with 700μl IP buffer, bound proteins were eluted in 15 μl 5x Laemmli sample buffer and analysed by Western blot.