Overexpression of PKC-ζ inhibits insulin-stimulated tyrosine phosphorylation of IRS-4. NIH-3T3IR cells were transiently cotransfected with HA-tagged IRS-1 or myc-tagged IRS-4 and either an empty control vector or PKC-ζ. Transfected cells were treated with 100 nm insulin for 0, 2, or 60 min. A, Experiments were as described in Fig. 3A. B, Cell lysates were immunoprecipitated (IP) with anti-myc antibody and subsequently immunoblotted (IB) with antiphosphotyrosine (top panel) or anti-IRS-4 antibody (bottom panel). Results from five independent experiments were quantified by scanning densitometry and expressed as mean ± sem of tyrosine-phosphorylated IRS protein normalized to total IRS protein. Overexpression of PKC-ζ significantly reduced insulin-stimulated tyrosine phosphorylation of both IRS-1 and IRS-4 at 2 and 60 min when compared with control cells (*, P = 0.04; §, P = 0.04; ¶, P = 0.01; #, P = 0.04).