Western blot showing Csk coimmunoprecipitation by the two HA-tagged PTPN22 proteins (R620 and W620) expressed in 293T cells. W620 decreases the affinity of PTPN22 for Csk. Increasing the amount of PTPN22 W620 expressed does not increase the amount of Csk that coimmunoprecipitates. Quantitation of Csk band intensity indicates a 2.9-fold difference between R620 and W620. Similar results (2.6-fold) were seen in a second experiment. PTPN22 sequences were cloned by PCR and verified to encode a PTPN22 protein sequence corresponding to Swiss-Prot Q9Y2R2. The R620W variant was introduced (QuikChange [Stratagene]), and both PTPN22 variants with an N-terminal HA tag were cloned into the pCMV5 expression vector (Qbiogene). Csk was cloned by PCR, the sequence was verified, and it was introduced into the pcDNA-DEST40 expression vector (Invitrogen). The 293T (GenHunter) cells (3×105 cells/well) were seeded in 12-well plates and were transfected 24 h later by use of 3.5 μl/well of Lipofectamine 2000 (Invitrogen). Cells were washed and resuspended in lysis buffer (50 mM Tris [pH 8.0], 2 mM EDTA, 1% NP-40, 50 mM NaF, 1 mM sodium orthovanadate, and 1× protease inhibitor cocktail [Sigma]) 48 h after transfection. HA-PTPN22 immunoprecipitations were performed as described elsewhere (Cloutier and Veillette ), with the following modifications. Before immunoprecipatation, detergent-insoluble material was removed by centrifugation (100,000×g) and lysates were pre-cleared by use of mouse IgG agarose beads (Sigma). The lysate (100 μg) was incubated with 15 μl of anti-HA conjugated beads (Sigma) for 2 h at 4°C. Beads were washed three times in wash buffer (50 mM Tris [pH 8.0], 100 mM NaCl, 2 mM EDTA, 1% NP-40, 50 mM NaF, and 1 mM sodium orthovanadate). Precipitated proteins and lysates were analyzed by western blot, by use of anti-HA (Covance) and anti-Csk (Upstate 06-566) antibodies, and were detected by use of HRP-conjugated secondary antibodies (Pierce 31430 and Biosource ALI3404) and chemiluminescence (Pierce).