Fig. 2.

Detection of EGF–dependent reversible oxidation of PTPs in IMR90 cells. IMR90 fibroblasts were cultured to confluency in low-glucose DMEM supplemented with 10% fetal bovine serum and nonessential amino acids. On the day before the experiment, cells were serum-deprived in low-glucose DMEM without phenol red for a total of 16 hours. Healthy cells were then incubated with 100 ng/ml EGF for 2 min and transferred to an argon-equilibrated hypoxic chamber through the airlock, and the cysteinyl-labeling assay was performed. Proteins were detected with biotin-HRP, RPTPα, SHP-2, or PTP1B (FG6) antibodies. Proteins were then detected with HRP-conjugated secondary antibodies, and the immunoreactive bands were visualized by ECL. Reversible oxidation of RPTPα, SHP-2, and PTP1B was observed upon 2 min of EGF stimulation in IMR90 cells. IAP labeling detected the reversible oxidation of PTPs specifically because no immunoreactive band was visualized when TCEP was omitted from the reducing step. Probing for biotinylated PTPs with biotin-HRP revealed an increase in staining of several bands, confirming the successful enrichment of multiple reversibly oxidized proteins. PD, Pull-down; WB, Western Blot.