Additionally, one set of samples was pretreated with vehicle Dorsomorphin research buy or 10 mM dimedone for 1 h prior to stimulation and sulfenic acid labeling. For immunoprecipitation experiments, 2–4 × 106 purified B cells were stimulated with 10 μg/mL anti-IgM and lysates were prepared as previously described by Michalek et al.

[14]. Briefly, cells were washed with PBS prior to lysis in the presence of DCP-Bio1 and lysates were precleared for 1 h at 4°C with protein G beads (Dynal). Following magnetic bead removal, lysates were incubated with 2.5 μg/mL anti-SHP-2 (BD Pharmingen), anti-SHP-1, or anti-actin (Santa Cruz Biotechnology) at 4°C with constant rotation overnight. The following day, protein G beads were added and the lysates were rotated at 4°C for 3 h. After discarding the supernatant, the magnetic beads were washed three times, resuspended in lysis buffer, and protein was eluted by boiling in reducing sample buffer (Pierce). Affinity capture of biotinylated proteins was performed according to Nelson et al. [47]. Samples were boiled with reducing sample buffer, separated on a 10% precast SDS denaturing gel, and transferred to a PVDF (polyvinylidene fluoride) membrane. The selleck chemicals llc membrane was blocked and probed with anti-PTEN (Cell Signaling) or anti-CD45 (Santa Cruz Biotechnology) according to the manufacturer’s protocol. For sulfenic acid

detection, samples lysed in the presence of 1 mM dimedone were separated on a 10–12% precast SDS denaturing gel and transferred to a PVDF membrane. The membrane was blocked and incubated with anti-dimedone antibody (Millipore) according to the manufacturer’s protocol. Proteins were visualized as previously described [14]. The blot was stripped and probed with anti-actin.

To quantify sulfenic acid, actin and cysteine sulfenic acid levels were normalized between samples using a Kodak Image Station 4000R and Carestream Protirelin Molecular Imaging Software. The entire length of the gel lane was used to determine sulfenic acid levels. Only the protein band was used for actin. The sulfenic acid signal was then normalized to actin protein levels. Detection of sulfenic acid during immunoprecipitation experiments was performed as previously described [14]. Briefly, samples lysed in the presence of 5 mM DCP-Bio1 were separated on a 7.5–15% SDS denaturing gel and transferred to a PVDF membrane. The membrane was blocked overnight at 4°C with 5% FCS in tris buffered saline supplemented with 0.1% Tween-20 (TBS-T). The following day, the membrane was washed three times and incubated with 1:50,000 dilution Streptavidin-HRP (Southern Biotech) in 5% FCS in TBS-T for 1 h at room temperature. After washing, the membrane was developed as previously described [14]. CFSE (5–6-carboxyfluorescein diacetate, succinimidyl ester, Molecular Probes) was resuspended in DMSO at a 5 mM stock and stored at −20°C. Purified B cells were washed with cold PBS three times and resuspended in PBS at 20 × 106 cells/mL. The CFSE stock solution was diluted in PBS to 6.