Cid 755673

HEK293 cells were grown in Dulbecco's modified eagle medium (DMEM) supplemented with 10% (v/v) Fetal Bovine serum (GE Healthcare, Little Chalfont, UK), 2 mm glutaMAX (ThermoFisher Scientific, Waltham, MA, USA), 100U·mL⁻¹ Penicillin and 100 μg·mL⁻¹ Streptomycin (ThermoFisher Scientific). Anti‐GST, Anti‐FLAG M2 antibody, HA antibody and agarose resins were purchased from Sigma (St. Louis, MO, USA). Glutathione sepharose 4B beads were from GE healthcare. Anti‐phosphotyrosine antibody (4G10) was from Millipore (Billerica, MA, USA), PKD anti‐pSer‐744/748 antibody, anti‐PKCδ antibody, secondary HRP‐linked goat anti‐Rabbit and Horse, anti‐Mouse antibodies were from Cell Signaling Technologies (Beverly, MA, USA). An in‐house site‐specific phospho‐Tyr antibody targeting pTyr in the P + 1 loop (CPApYLAPEV), which is cross‐reactive between PKD isoforms due to 100% homology of the epitope is described previously 36. Phorbol 12,13‐dibutyrate (PDB), ATP, STI‐571, PP2, CRT 0066101, CID 755673 and Hydrogen peroxide 30% (v/v) were from Sigma, Polyethyleneimine (PEI) was from Polysciences Inc. (Warrington, PA, USA).

All experiments were performed using Chinese Hamster ovary cells, CHO-K1 (Cat. No 85051005; European Collection of Cell Cultures, Salisbury, UK), which do not express endogenous EGFR. Cells were cultured in Ham's F-12 Nutrient Mixture (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (Life Technologies, Carlsbad, CA) and 50 μg/ml gentamycin (Life Technologies) at 37°C under 5% CO₂ atmosphere. Two days prior to imaging, cells were seeded on a glass bottom 8-well Lab-Tek chamber slide (Thermo Scientific, Rockford, IL) and transfected with one or combination of following plasmids: eGFP/pcDNA3, Lyn-eGFP/pcDNA3, Lyn-mCherry/pcDNA3, wild type or mutant eGFP-EGFR/pcDNA3, mCherry-HA-PDK1/pcDNA3, PLCγ1-mCherry/pcDNA3, pEKAREV [46 (link)] (~0.2 μg of each plasmid per well) using FuGENE6 transfection reagent (Promega, Madison, WI) according to the manufacturer’s protocol. The EGFR expression level was evaluated by the confocal microscopy. Before imaging, cells were incubated in serum-free F-12 for ≥ 20 min for serum starvation. For PKC activation experiments, cells were incubated for ≥ 20 min in serum-free F-12 containing of 200 nM phorbol 12-myristate 13-acetate (PMA; InvivoGen, Toulouse, France) and 0.02% Kolliphor EL (Sigma-Aldrich). For PKD inhibition experiments, cells were incubated with 10 μM CID755673 (Sigma-Aldrich) in the presence of 0.02% Kolliphor EL.