Anti flag m2 and anti ha antibodies

HCT116 cells continuously expressing FLAG-HA-SMYD3 were generated by stable transfection of pIRES-FLAG-HA-SMYD3. Nuclear extracts were prepared as described (30 (link)), and ectopic SMYD3 and its interacting proteins were purified by sequential immunoprecipitation using anti-FLAG M2 and anti-HA antibodies (Sigma) in the precipitation buffer (20 mM Tris-HCl, pH 7.3, 300 mM KCl, 0.2 mM EDTA, 20% glycerol and 0.1% Nonidet P-40). The purified proteins were resolved by 4–20% gradient SDS-PAGE, and stained with Coomassie blue. Major protein bands were excised and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

The following antibodies were raised by the Division of Signal Transduction Therapy at the University of Dundee in sheep and purified against the relevant antigens: anti‐CDKL5 (S957D; raised against amino acids 350–650 of human CDKL5 expressed in bacteria using plasmid DU50406; third bleed); anti‐MAP1S phospho‐Ser⁹⁰⁰ (SA339; raised against the peptide KKRASRPLpS⁹⁰⁰ARSEPSE conjugated to bovine serum albumin; third bleed); anti‐CEP131 phospho‐Ser³⁵ (SA373; raised against the peptide CKKPPVSRRPGpS³⁵AATTKP conjugated to bovine serum albumin; third bleed); anti‐CDKL5 phospho‐Tyr¹⁷¹ (SA547; raised against the peptide GNNANYTEpY¹⁷¹VATRWYR conjugated to bovine serum albumin; third bleed). Sheep were immunised with each antigen followed by up to five further injections 28 days apart, with bleeds performed 7 days after each injection.
Anti‐FLAG (M2) and anti‐HA antibodies were obtained from Sigma, and anti‐GAPDH (14C10) antibodies were purchased from Cell Signalling. Secondary anti‐sheep, anti‐mouse and anti‐rabbit HRP‐conjugated antibodies were purchased from Life Technologies and were used at 1:5,000–1:10,000 dilutions, for 1 h at room temperature. Anti‐pTyr antibodies (4G10) were from Merck.