Fig 2: Y1150-biased phosphorylation in the IR kinase domain by IR-A48. (A) The phosphorylation of six tyrosine residues of IR was analyzed using specific phospho-antibodies. Rat-1/hIR cells were stimulated with 200 nmol/l IR-A48 and 50 nmol/l insulin for 10 min. (B) Specificity of the phospho-antibodies 10C3 and pAb for mono-pY1150, mono-pY1151 and dual-pY1150/pY1151 peptides. 10C3 is a monoclonal antibody purchased from Santa Cruz (sc-81500) and pAb is a polyclonal antibody purchased from Invitrogen (44804G). Data represent the mean ± S.E. of four biological replicates and similar results were obtained from two independent experiments. Black bar, 10C3; white bar, pAb. (C) Non-cross-reactivity of IR-A48 with IGF-1R. Following incubation of HeLa cells with 50 nmol/l IGF-1, 100 nmol/l insulin and 1 µmol/l IR-A48 for 1 h, IGF-1R was immunoprecipitated. The monoclonal antibody 10C3 was used to detect pY1135 of IGF-1R.