Pkc lipid activator

After incubation of recombinant PHB2 (Abnova, H00011331-P01) with recombinant PKCα (Merck Millipore, 14–484), PKC lipid activator (Merck Millipore, 20–133) and 0.5 mM ATP (Takara, 4041) for 30 min at 30 °C, the reaction samples were incubated with anti-phospho-PHB2 (S39) antibody at 4 °C for 12 h. The complexes were then precipitated using rec-Protein G Sepharose 4B at 4 °C for 1 h. After washing three times with lysis buffer, the purification of full-length PHB2 with phosphorylation at S39 was verified using immunoblot analysis.

c-Abl was precipitated from 500 μg whole cell lysates using 3 μg anti-c-Abl (AB3, Merck Biosciences, Germany). The in vitro kinase reaction was performed in 20 mM HEPES pH 7.4, 10 mM MgCl₂, 10 mM MnCl₂, 250 μM ATP using 250 ng purified GST-Crk aa 120–225 [26 (link)] for 30 min at 30 °C. PKC-mediated c-Abl phosphorylation was performed using 10 ng/μl recombinant PKCαβγ (Merck Millipore, Germany), 100 ng/μl recombinant c-Abl (Merck Millipore, Germany) and 250 μM ATP for 10 min at 30 °C in an assay dilution buffer II reaction buffer (Merck Millipore, Germany). To activate PKC activity, a PKC lipid activator (Merck Millipore, Germany) has been added to the reaction as recommend by manufacturer’s instructions. TAP pull-downs were performed using the Interplay Mammalian TAP System (Agilent Technologies, Austria) according to the manufacturer’s manual.

Recombinantly expressed and purified human FLNc d18–21 was dialyzed overnight at 4 °C in dialysis buffer (1 mM DTT, 100 mM KCl, 20 mM HEPES pH 7.4, 10 mM MgCl₂). For MS-coupled kinase assays, H₂O was added to 100 μg protein to a total volume of 200 μl and mixed with 10× kinase buffer (NEB, Frankfurt, Germany). The assay was started by adding 200 ng Akt (Proteinkinase, Kassel, Germany) and/or 200 ng PKCα (Sigma-Aldrich) in the presence of 1× PKC lipid activator (Merck Millipore, Darmstadt, Germany). The reaction was carried out for 20 min at 30 °C and 200 rpm. One tenth of each of three independent replicates was used for analysis by SDS-PAGE and immunoblotting using an antibody directed against the EEF-tag fused carboxy-terminally to FLNc d18–21. The remaining sample was diluted 1:4 (v/v) with 50 mM ammonium bicarbonate and subjected to in-solution digestion using sequencing grade trypsin (1:50) (Promega) for 3.5 h at 42 °C and 200 rpm on a thermoshaker. Single protein digests were acidified with TFA [final concentration 1% (v/v)]. Phosphopeptides were enriched using TiO₂ beads and analyzed by LC-MS applying a 1 h LC gradient and MSA, HCD or electron transfer dissociation (ETD) for peptide fragmentation. The activation time for ETD fragmentation was set to 100 ms. using MSA, HCD and ETD fragmentation.