Isolation and Analysis of Cellular Fractions

Nuclear and cytoplasmic extracts from naive T cells cultured for 3 days were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce), as previously described^{21 (link)}. The purity of the nuclear and cytoplasmic fractions was verified by probing with antibodies against Lamin B1 (D4Q4Z; 1,000×; Cell Signaling Technology). Whole-cell extracts were immunoprecipitated with an anti-Raptor (24C12; 100×; Cell Signaling Technology), anti-Def6 (Rabbit polyclonal; 100×^{21 (link)}), anti-p62 (H-290; 50×; Santa Cruz), anti-TRAF6 (H274; 50×; Santa Cruz), or anti-HA (3F10; 50×; Roche Applied Science) antibodies. Antibodies to p-STAT3 (Y705; 1,000×), p-4E-BP (T37/46; 1,000×), 4E-BP (1,000×), p-S6K1 (S371; 1,000×), S6K1 (1,000×), p-AKT (S473; 1,000×), AKT (1,000×), p-AKT (T308; 1,000×), p-PRAS40 (T246; 1,000×), PRAS-40 (1,000×), p-AMPK (T172; 1,000×), AMPK (1,000×), p-Raptor (S792; 1,000×) and p62 (5114; 1,000×) were obtained from Cell Signaling Technology. Antibodies to IRF4 (M-17; 1,000×), TRAF6 (H274; 500×), and c-Myc (9E10; 500×) were obtained from Santa Cruz. Anti-Bcl6 antibody was obtained from BD (K112-91; 1,000×). Anti-Flag monoclonal antibody M2 (horseradish peroxidase (HRP)) was obtained from Sigma (1,000×).

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Yi W., Gupta S., Ricker E., Manni M., Jessberger R., Chinenov Y., Molina H, & Pernis A.B. (2017). The mTORC1-4E-BP-eIF4E axis controls de novo Bcl6 protein synthesis in T cells and systemic autoimmunity. Nature Communications, 8, 254.

Publication 2017

Lamin B1 (D4Q4Z; anti-Raptor (24C12; anti-TRAF6 (H274 anti-HA (3F10 p-STAT3 p-4E-BP p-S6K1 p-AKT p-PRAS40 PRAS-40 p-AMPK p-Raptor TRAF6 (H274 c-Myc (9E10; Anti-Bcl6 antibody

Anti antibody Antibodies Bcl6 C myc Cell extracts Cytoplasmic Horseradish peroxidase Irf4 Lamin b1 Pras Rabbit Raptor Stat3 Traf6

Corresponding Organization :

Other organizations : Hospital for Special Surgery, Cornell University, TU Dresden, Rockefeller University

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Variable analysis

independent variables

Nuclear and cytoplasmic extracts from naive T cells cultured for 3 days

dependent variables

Protein levels or modification (phosphorylation) of Lamin B1, Raptor, Def6, p62, TRAF6, STAT3, 4E-BP, S6K1, AKT, PRAS40, AMPK, Raptor, p62, IRF4, TRAF6, c-Myc, Bcl6

control variables

Whole-cell extracts from naive T cells cultured for 3 days

positive controls

Antibodies against Lamin B1 to verify the purity of nuclear and cytoplasmic fractions

negative controls

No negative controls were explicitly mentioned in the input protocol.

Annotations

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