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1.
Fig. 3.

Fig. 3. From: Tripartite motif containing protein 27 negatively regulates CD4 T cells by ubiquitinating and inhibiting the class II PI3K-C2?.

TRIM27 mediates K48 polyubiquitination of PI3KC2β. GFP-TRIM27 was cotransfected with FLAG-PI3KC2β and ubiquitin. FLAG-PI3KC2β was then immunoprecipitated with anti-FLAG antibodies and Western blotted with antibodies that recognize various ubiquitin modifications as indicated.

Xinjiang Cai, et al. Proc Natl Acad Sci U S A. 2011 December 13;108(50):20072-20077.
2.
Fig. 1.

Fig. 1. From: Tripartite motif containing protein 27 negatively regulates CD4 T cells by ubiquitinating and inhibiting the class II PI3K-C2?.

TRIM27 and PI3KC2β associate in vivo. (A) FLAG-TRIM27 was cotransfected with GFP-PI3KC2β or GFP-PI3KC2α in HEK293 cells and the ability of the two proteins to coimmunoprecipitate was assessed as indicated. (B) GFP-TRIM27 was cotransfected with mCherry-PI3KC2β in COS cells and colocalization was assessed by fluorescence. Magnification, 60×. (C) Lysates from 293 cells were immunoprecipitated with anti-PI3KC2β or a control antibody and then immunoblotted with anti-TRIM27 or anti-PI3KC2β antibodies.

Xinjiang Cai, et al. Proc Natl Acad Sci U S A. 2011 December 13;108(50):20072-20077.
3.
Fig. 6.

Fig. 6. From: Tripartite motif containing protein 27 negatively regulates CD4 T cells by ubiquitinating and inhibiting the class II PI3K-C2?.

Increased cytokine production by TRIM27−/− Th0 cells. TRIM27−/− and TRIM27+/+ CD4 T cells were stimulated for 3 d with antiCD3/CD28 antibodies and, after resting overnight, were restimulated with irradiated splenocytes at a ratio of 1:8 together with various concentrations of SEE. Cytokines were assessed in supernatants as indicated 72 h after stimulation. (A) IFN-γ, (B) IL-2, and (C) TNF-α. Plotted are means ± SE and are representative of two independent experiments performed in quadruplicates.

Xinjiang Cai, et al. Proc Natl Acad Sci U S A. 2011 December 13;108(50):20072-20077.
4.
Fig. 2.

Fig. 2. From: Tripartite motif containing protein 27 negatively regulates CD4 T cells by ubiquitinating and inhibiting the class II PI3K-C2?.

TRIM27 ubiquitinates PI3K-C2β. (A) FLAG-TRIM27(WT) or a FLAG-TRIM27 RING MT were cotransfected with GFP-PI3KC2β or GFP-PI3KC2β kinase dead (KD) together with His6-tagged ubiquitin in 293 cells as indicated. Following lysis, ubiquitinated proteins were purified using Ni-NTA agarose and ubiquitinated GFP-PI3KC2β was then quantitated by Western blot with anti-GFP antibodies. (B) Shown is the inability of TRIM27 to stimulate the ubiquitination of GFP-PI3KC2α using the protocol described in A. (C) 293 Cells were cotransfected with GFP-PI3KC2β and His6-tagged ubiquitin together with an siRNA to TRIM27 (siRNA-1, -2) or a control siRNA and ubiquitinated-GFP-PI3KC2β was assessed as described in A. siRNA-2 targets a sequence in the 3′ UTR of the endogenous TRIM27. siRNA-2–transfected cells were rescued by transfecting a TRIM27 full-length cDNA lacking the 3′ UTR.

Xinjiang Cai, et al. Proc Natl Acad Sci U S A. 2011 December 13;108(50):20072-20077.
5.
Fig. 5.

Fig. 5. From: Tripartite motif containing protein 27 negatively regulates CD4 T cells by ubiquitinating and inhibiting the class II PI3K-C2?.

KCa3.1 channel activity and TCR-stimulated Ca2+ influx is increased in TRIM27−/− CD4 Th1 and Th2 cells. (A) Real-time PCR of GATA-3 and T-bet expression in Th1 and Th2 differentiated cells. Bar graph summary of whole-cell patch-clamp experiments performed on (Bi) Th1 and (Ci) Th2 cells using a protocol similar to that described in Materials and Methods and Fig. S1 (n = 15 cells). Shown is the TRAM34 (KCa3.1) and Shk (Kv) sensitive current at +60 mV. *P < 0.005 for increase in TRAM-34 sensitive current between TRIM27−/− and WT cells. Anti-CD3 stimulated Ca2+ flux was assessed on (Bii) Th1 and (Cii) Th2 cells as described in Fig. 6A. All experiments shown are representative of at least three experiments performed on cells isolated from at least three separate mice.

Xinjiang Cai, et al. Proc Natl Acad Sci U S A. 2011 December 13;108(50):20072-20077.
6.
Fig. 4.

Fig. 4. From: Tripartite motif containing protein 27 negatively regulates CD4 T cells by ubiquitinating and inhibiting the class II PI3K-C2?.

Ubiquitination of PI3KC2β inhibits PI3KC2β‘s enzymatic activity and does not stimulate PI3KC2β degradation. (A) FLAG-TRIM27(WT) or RING MT were transfected into 293 cells and levels of endogenous PI3KC2β was then assessed in by Western blot in cells that were either untreated or treated with the proteosome inhibitor MG132 48 h after transfection. (B) Jurkat T cells were stimulated with anti-CD3/CD28 antibodies and protein levels of endogenous TRIM27 and PI3KC2β were assessed by Western blot at various times after stimulation. (C) GFP-PI3KC2β (WT) or GFP-PI3KC2β (KD) were cotransfected with TRIM27 (WT) or TRIM27 (RING MT), and PI3KC2β kinase activity was determined on anti-GFP immunoprecipitates, as previously described by the authors (19). Shown (Lower) is an anti-GFP Western blot of lysates from transfected cells demonstrating equal levels of GFP-PI3KC2β expression. Plotted are means ± SE from two independent experiments performed in triplicate. *P < 0.01.

Xinjiang Cai, et al. Proc Natl Acad Sci U S A. 2011 December 13;108(50):20072-20077.

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