FIG. 4.
Insulin-induced tyrosine phosphorylation in TCPTP−/− versus TCPTP+/+ cells. (A) TCPTP−/− (EFM4−/−) or TCPTP+/+ (EFM7+/+) cells were either left untreated or stimulated with 100 nM insulin (INS) for 10 min. Cells were lysed, and proteins were resolved on SDS-PAGE and immunoblotted with phosphotyrosine (pTyr)-specific antibodies. Molecular mass standards (precision prestained standards; Bio-Rad, Hercules, Calif.) are indicated on the right, and an arrow on the left highlights the ≈95-kDa pTyr protein in TCPTP−/− cells following insulin stimulation. (B) TCPTP−/− (EFM4−/−) or TCPTP+/+ (EFM7+/+) cells were serum starved and either left untreated or stimulated with 10 nM insulin for 60 min. Equal amounts of protein were resolved by SDS-PAGE and immunoblotted with antibodies specific for pTyr and then stripped and reprobed with antibodies specific for the IR β-subunit (IRβ). (C) EFM14−/− or EFM11+/+ cells were serum starved and either left untreated or stimulated with 10 nM insulin for 60 min. Proteins were resolved and immunoblotted for pTyr and then stripped and reprobed for IR β-subunit. Molecular mass standards (prestained protein markers, broad range; New England Biolabs, Beverly, Mass.) are indicated on the right, and an arrow highlights the ≈95-kDa pTyr protein in TCPTP−/− cells following insulin stimulation. (D) TCPTP−/− (EFM4−/−) or TCPTP+/+ (EFM7+/+) cells were serum starved and either left untreated or stimulated with 10 nM insulin for 60 min. Cells were lysed in immunoprecipitation lysis buffer, and the pTyr-containing proteins were immunoprecipitated as described under Materials and Methods. The immunoprecipitates (IPs) and equal quantities of the corresponding lysates prior to immunoprecipitation were resolved by SDS-PAGE and immunoblotted with antibodies specific for IRβ. Representative immunoblots are shown for clone 45-R5 in response to 1 or 10 nM insulin. Quantitations of PKB/Akt phosphorylation in response to insulin stimulation in TCPTP−/−, 45-R5, 45-R19, and 45-R20 cells are also shown. In each case the phospho-Akt immunoblots were quantitated by densitometric analysis and normalized for total Akt protein in corresponding Akt immunoblots, with the phospho-Akt/Akt ratio in the absence of insulin being set at zero. Units are arbitrary and are means ± standard errors of at least three independent experiments.