Fig 2: DEP-1 protein expression is reduced in the liver after antisense oligonucleotide (ASO) treatment. A: DEP-1 protein expression was analyzed after wheat-germ-agglutinin (WGA) precipitation and visualized by immunoblotting of tissues derived from control ASO and DEP-1 ASO treated mice; (n = 4 per group). B: Densitometric analysis of immunoblotting data was performed after normalization to the housekeeping protein GAPDH and expressed as arbitrary units; (n = 8–9 per group). **P < 0.01.

Fig 3: PTPRJ suppresses leptin signaling through the dephosphorylation of Y813 and Y868 in JAK2. (A) Suppression of the tyrosyl phosphorylation of LepRb and JAK2 by the co-expression of PTPRJ in HEK293T cells. FLAG-LepRb and JAK2 expression constructs were co-transfected with an empty vector or HA-PTPRJ expression construct. Cells were treated with/without 50 ng/ml leptin for 15 min, and LepRb proteins were immunoprecipitated with an anti-FLAG antibody and analyzed with the indicated antibodies. The expression and phosphorylation of JAK2 in lysates were analyzed with anti-JAK2 and phospho-JAK2 (pY1007/1008) antibodies. PTPRJ proteins were detected with an anti-HA antibody. The right two graphs show summaries of the phosphorylation levels of LepRb and JAK2 (n = 4). Full-length blots are presented in Supplementary Fig. S9. (B) Dephosphorylation assays using synthetic phosphopeptides. Schematic representation of phosphorylation sites in LepRb and JAK2 (left). CRH1; cytokine receptor homology 1: CRH2; cytokine receptor homology 2: Ig-like; immunoglobulin-like: FN-III, fibronectin type III repeat: Box 1; box 1 motif: FERM; four-point-one, ezrin, radixin, moesin domain: SH2; src homology 2. List of phosphopeptides (middle). Summary of phosphatase assays (right). Phosphopeptides were incubated with GST-PTPRJ, and the amount of phosphate released was measured (n = 3). (C) Tyrosine phosphorylation levels of wild-type and mutant JAK2 with or without PTPRJ expression. The right graph shows a summary of the phosphorylation levels of JAK2 (n = 4). Full-length blots are presented in Supplementary Fig. S10. (D) Suppression of the SH2B1-enhanced phosphorylation of LepRb and JAK2 by the co-expression of PTPRJ in HEK293T cells. An mCherry-SH2B1 expression construct was co-transfected with the indicated constructs. The right two graphs show summaries of the phosphorylation levels of LepRb and JAK2 (n = 4 each). Full-length blots are presented in Supplementary Fig. S11. The graphs of (A) and (D) show relative values to that without the leptin stimulation, and the graph of (C) shows relative values to that with the leptin stimulation. All data are expressed as the mean ± s.e.m. *P < 0.05 and **P < 0.01 represent significant differences between the indicated pairs (ANOVA followed by Scheff’s post-hoc test).

Fig 4: Metabolic phenotyping of wild-type and Ptprj-/- mice. (A) Body weight of mice was determined twice weekly over 8 weeks. (B,C) ITT was performed after 4 h fasting and the AUC was calculated. (D,E) GTT was conducted after 12 h fasting and corresponding AUC was calculated (n = 8–10 mice per genotype). LFD WT vs. LFD Ptprj KO *p < 0.05; HFD WT vs. HFD Ptprj KO †p < 0.05. (F) Respiratory exchange ratio determined over 24 h and (G) mean of data recorded every 15 min (n = 6–10 mice per genotype). LFD WT vs. LFD Ptprj KO *p < 0.05, ***p < 0.001; HFD WT vs. HFD Ptprj KO †p < 0.05, †††p < 0.001.

Fig 5: DEP-1 expression and genotyping of wild-type and Ptprj-/- mice. (A) DEP-1 expression based on activity measurements under reduced conditions (as outlined in the Materials and Methods section) in metabolic tissues derived from wild-type mice. (B) Wild-type and Ptprj-/- mice were characterized by PCR. (C, D) Confirmation of DEP-1 knockout in liver tissue visualized by immunoblotting and DEP-1 activity measurements (n = 3–4 mice per genotype).