Dissecting EGF Signaling Regulation

Anti-CAR (H300), anti-HSC70 and anti-PKCδ antibodies were from Santa Cruz Technology. p-CAR Thr²⁹⁰/Ser²⁹³ polyclonal antibody was previously described (7 (link)) and was developed by Perbioscience (Thermofisher) using the peptide Ac- RTS(pT)AR(pS)YIGSNH-C and was affinity purified before use. Anti-β-tubulin, anti- acetylated tubulin and anti-FLAG antibodies were from Sigma Aldrich. Anti-phospho-EGFR (Y1173), anti-phospho-ERK (T202/Y204), anti-EGFR and anti-ERK antibodies were from Cell Signalling. Anti-KIF22 antibody was from Genetex, anti-GAPDH was from Chemicon. Anti-GFP antibodies were from Roche (immunoblotting) and MBL (immunoprecipitation). Anti-Ki67 and anti-phospho H3 antibodies were from Leica. Anti-Halo-tag antibody and HaloTag™ direct ligand were from Promega. Anti-mouse HRP and anti-rabbit-HRP were from DAKO. Anti-mouse-568, anti-rabbit-568 and phalloidin-647 were all obtained from Invitrogen. Recombinant human EGF was acquired from Peprotech and CalyculinA, sodium orthovanadate and protease inhibitor cocktail 1 were obtained from Calbiochem. Nocodazole and Dynasore were from Sigma. KIF22 targeted siRNA were from Origene and CAR and non-targeting siRNA were acquired from Dharmacon. PKCδ targeting siRNA were from Ambion.