Fig 2: Alternatively spliced forms of human PTPN22. A. Schematic diagrams of the human ptptn22 gene and putative protein products of each isoform. Exons are numbered and white boxes represent un-translated exons. The yellow box represents protein tyrosine phosphatase (PTP) domain. The key catalytic region (HCSAGC) and R620 are marked. The silver box after the PTP domain is the inhibitory domain. The red boxes represent proline rich regions. The brown two-headed arrows indicate the amplification products of real-time PCR for each isoform. The exons constituting each isoform are indicated. B. 293T cells were transfected with an expression vector expressing indicated FLAG-PTPN22 isoforms. The protein levels of FLAG-PTPN22 isoforms and Hsp90 in the transfected cells were determined with Western blotting. The data shown is representative of three independent experiments. C. cDNA was prepared from human CD4+ T cells, which were stimulated in vitro with anti-CD3/anti-CD28 for indicated periods of time, and subjected to real-time PCR analysis with isoform-specific primers. The transcript levels thus obtained were normalized against those of ß-actin from the same samples. The data shown are the results from three healthy donors.

Fig 3: PTPN22.6 is a dominant negative variant of PTPN22.1. A. Jurkat cells were transfected with a NFAT-luc reporter, 10 µg of pCMV2B expressing indicated FLAG-PTPN22 isoforms, and a TK-Renilla reporter. The transfected cells were then stimulated with anti-CD3 overnight and the luciferase activity was analyzed. Normalized firefly lucifease activity was calculated as described in Methods and was shown. The data shown are cumulative results of at least three independent experiments. B. Jurkat cells were transfected with the NFAT-luc, TK-Renilla, 10 µg of pCMV2B-FLAG-PTPN22.1, and increasing amount of pCMV2B-FLAG-PTPN22.6. Empty pCMV2B vector was added to equalize the total amount of expression vector in each sample. Normalized luciferase activity was shown. The data shown in the top panel are cumulative results of at least three independent experiments. The standard error bars are too narrow to see. pCMV2B-FLAG-PTPN22.1 and pCMV2B-FLAG-PTPN22.6 at the same ratio was used to transfect 293T cells. The protein level of FLAG-PTPN22.1 and FLAG-PTPN22.6 in transfected 293T cells were quantified with Western blotting using FLAG antibody. One representative Western blot is shown. C. Jurkat cells were transfected with NFAT-luc, TK-Renilla, and increasing amount of the pCMV2B vector expressing indicated FLAG-PTPN22 protein. Transfected cells were stimulated and normalized luciferase activity was shown. The data shown are cumulative results of at least three independent experiments. *p<0.05. In all luciferase experiments shown in A, B, and C, the normalized luciferase activity obtained from cells transfected with empty pCMV2B and stimulated with anti-CD3 was arbitrarily set as 100. D. Primary human CD4+ T cells were transfected with indicated amount (in µg) of pCMV2B vector expressing indicated FLAG-PTPN22 variants. In some transfections, empty pCMV2B vector was added to bring up the total amount of expression vector to 5 µg. The transfected cells were stimulated with anit-CD3 (1 µg/ml) for 72 hours. The concentration of IL-2 in supernatant was measured with ELISA. The IL-2 level measured from cells transfected with empty pCMV2B vector (300–700 pg/ml) was arbitrarily set as 100%. Cell extract of the transfected cells was also probed with anti-FLAG and anti-Hsp90 antibodies in a Western blot to show the level of various FLAG-PTPN22 proteins. The data shown are cumulative results of at least three independent experiments. *p<0.05; **p<0.005. A representative Western blot is shown in the bottom panel.

Fig 4: Detection of endogenous PTPN22.6 protein in human T cells. A. Protein extract was prepared from HT-29 or Jurkat cells, which were mock (M) transfected or transfected with PTPN22 specific siRNA or scrambled (Sc) siRNA prior to harvest. The extract was analyzed with Western blotting using Lyp antibody. The arrowheads indicate protein species that are not present in HT-29 cells and are suppressed by PTPN22 siRNA. B. 293T cells were transfected with the plasmid vector expressing FLAG-PTPN22.1 or FLAG-PTPN22.6. The transfected cells were then analyzed with Western blotting using PTPN22.6 and Hsp90 antibody. C. Protein extract prepared from Jurkat cells described in A was subjected to immunoprecipitation with anti-Lyp antibody or control IgG. The immunoprecipitate along with protein extract prepared from 293T cells expressing FLAG-PTPN22.6 were analyzed with Western blotting using PTPN22.6 antibody. A fraction of Jurkat cell extract was set aside prior to immunoprecipitation and was probed with Hsp90 antibody to demonstrate equal input. IP and IB stand for immunoprecipitation and immunoblot, respectively. D. Primary human Th cells were harvested from healthy donors and expanded in vitro with anti-CD3/atni-CD28 for 5 days. The cells were then transfected with siRNA and subjected to immunoprecipitation as Jurkat cells described in A and C. The data shown in A, B, and C are representative of at least two independent experiments.

Fig 5: Expression of PTPN22 isoforms in RA patients.cDNA was prepared from whole blood of 41 RA patients described in Table 1. The cDNA was subjected to real-time PCR using PTPN22 isoform-specific primers. The transcript level thus obtained was normalized against that of ß-actin obtained from the same sample and plotted against DAS28-CRP3. Linear regression model was used to evaluate the relationship between the transcript level and DAS28-CRP3. The p value for slope estimate and R2 of each model were shown.