Nod1 associates with endogenous SGT1. (A) MCF-7 cells stably expressing empty vector or Nod1-FLAG were immunoprecipitated (IP) with anti-FLAG antibody, and coprecipitated proteins were revealed by immunoblotting using anti-SGT1 or anti-HSP90 antibodies. Expression of Nod1 was detected after stripping of membrane and staining with anti-FLAG antibody. Whole cell lysates (WCL) from both stable transfectants were checked for SGT1 expression. (B) 293 cells were transfected with empty vector or vectors encoding for indicated Nod1 deletion mutants. Cell lysates were immunoprecipitated with anti-Myc antibody, and coprecipitated proteins were revealed by immunoblotting using anti-SGT1 antibody. Expression of Nod1 wild type and mutants was detected after stripping of membrane and staining with anti-Myc antibody. WCL were checked for SGT1 expression. (C) 293 cells were cotransfected with Nod1-FLAG and indicated Myc-tagged SGT1 deletion mutants. Cell lysates were immunoprecipitated with anti-Myc antibody, and coprecipitated Nod1 protein was revealed by immunoblotting using anti-FLAG antibody. Expression of SGT1 wild type and mutants was detected after stripping of membrane and staining with anti-Myc antibody. WCL were checked for Nod1-FLAG expression. (D) 293 cells were transfected with empty vector or vectors encoding for various Myc-tagged NLR proteins. Cell lysates were immunoprecipitated with anti-Myc antibody, and coprecipitated proteins were revealed by immunoblotting using anti-SGT1 antibody. Expression of NLR proteins was detected after stripping of membrane and staining with anti-Myc antibody. WCL were checked for SGT1 expression.

SGT1 is a critical component of Nod1 signaling pathways. (A) MCF7 Nod1 cells were transfected with control siRNA or SGT1 no. 4 siRNA. Cells were stimulated with γTriDAP (20 μg/ml), IFN-γ (1,000 units/ml), or both for 2 h. Cell lysates were prepared, and JNK and STAT1 phosphorylation was analyzed by Western blotting using anti-phospho JNK and anti-phospho STAT1 antibodies. The same blots were reprobed for anti-JNK and anti-STAT1, indicating equal protein loading. Depletion of endogenous SGT1 protein was confirmed by Western blotting using anti-SGT1 antibody (bottommost panels). (B) MCF7 Nod2 cells were transfected with control siRNA or SGT1 no. 4 siRNA. Cells were stimulated with MDP (20 μg/ml) for 2 h or TNFα (10 ng/ml) for 30 min. Cell lysates were prepared, and JNK phosphorylation was analyzed by Western blotting using anti-phospho JNK antibody. The same blots were reprobed for anti-JNK, indicating equal protein loading. Depletion of endogenous SGT1 protein was confirmed by Western blotting using anti-SGT1 antibody (Bottom). (C–E) MCF7 Nod1 (C and E) and MCF-7 Nod2 (D) cells were transfected with control siRNA or siRNA targeting SGT1. Cells were left untreated or treated with γTriDAP or MDP (20 μg/ml each) in the presence or absence of IFN-γ (1,000 units/ml) for 36 h or TNFα (10 ng/ml) for 18 h. Cells were incubated with propidium iodide, and apoptotic cell death was measured by flow cytometry. Med, medium; Neg, negative.

Modulation of steady-state protein levels of Nod1 and other proteins. (A) Inhibition of HSP90 promotes degradation of Nod1. MCF-7 Nod1 or MCF-7 Nod2 cells were treated with GA (250 nM) for indicated times. Cells were lysed, and Nod1 or Nod2 expression was analyzed by Western blotting using anti-Myc antibody. The same blots were stripped and reprobed with indicated antibodies. (B) MCF-7 Nod1 or MCF-7 Nod2 cells were transfected with control siRNA or specific siRNAs targeting HSP90, SGT1, or Chp1. Depletion of the endogenous protein targeted by the siRNA was confirmed by Western blotting using indicated antibodies. The same blots were stripped and reprobed with specific antibodies noted in the figure. Neg, negative.

Impaired cytokine production in SGT1 depleted cells. (A and B) MCF-7 Nod1 (A) and MCF-7 Nod2 (B) cells were transfected with control siRNA or SGT1 no. 4 siRNA. Cells were left untreated or treated with γTriDAP or MDP (20 μg/ml each) in the presence or absence of IFN-γ (1,000 units/ml) for 24 h. Cell supernatants were harvested and assayed for IL-8 (Left) and MCP-1 (Right) production. (C) MCF-7 Nod1 cells were transfected with control or SGT1 no. 4 siRNA. Cells were treated with TNFα (10 ng/ml) for 24 h. Cell supernatants were harvested and assayed for IL-8 production. (D) Depletion of endogenous SGT1 protein using SGT1 no. 2 and no. 4 siRNAs was confirmed by Western blotting using anti-SGT1 antibody. The same blot was reprobed for anti-ERK2, indicating equal protein loading. Med, medium; Neg, negative.

Chp1 and SIP do not modulate Nod1 activities. (A and B) MCF-7 Nod1 were transfected with control siRNA or specific siRNAs targeting Chp1 or SIP. Depletion of endogenous Chp1 and SIP proteins was confirmed by immunoblotting using anti-Chp1 and anti-SIP antibodies. The same blots were reprobed for anti-ERK2, indicating equal protein loading. (C and D) MCF-7 Nod1 cells were transfected with control, Chp1 no. 4 (B), or SIP no. 2 (C) siRNAs. Cells were left untreated or treated with γTriDAP (20 μg/ml) in the presence or absence of IFN-γ (1,000 units/ml) for 24 h. Cell supernatants were harvested and assayed for IL-8 production. (E and F) MCF-7 Nod2 cells were transfected with control, Chp1 no. 4 (E), or SIP no. 2 (F) siRNAs. Cells were left untreated or treated with MDP (20 μg/ml) or TNFα (10 ng/ml) for 24 h. Cell supernatants were harvested and assayed for IL-8 production. Med, medium; Neg, negative.