1. Volume 9, Issue 1, Pages 121-131 (January 2002)
A Mammalian Homolog of Drosophila schnurri, KRC, Regulates TNF Receptor-Driven Responses and Interacts with TRAF2 
Mohamed Oukka, Sean T. Kim, Geancarlo Lugo, Jenny Sun, Lai-Chu Wu, Laurie H. Glimcher 
Molecular Cell 
Volume 9, Issue 1, Pages (January 2002)
DOI: /S (01)

2. Figure 1 KRC Is a Negative Regulator of Endogenous TNFα Expression
Northern blotting analysis was performed using total RNA made from RAW cell lines transfected with an empty vector as a control and from a panel of nine independent RAW clones stably transfected with full-length KRC (A) and three RAW clones stably transfected with dominant-negative KRC (B). The blot was probed with a TNFα cDNA and with HPRT as loading control.
Molecular Cell 2002 9, DOI: ( /S (01) )

3. Figure 2
KRC and KRCtr Inhibit while Antisense and Dominant-Negative KRC Increase TNFα-Driven NFκB Transactivation
293T cells (3 × 105 ) were transfected with 25 ng of NFκB luciferase reporter plasmid, 50 ng of CMVβ-gal, and 1 μg of each indicated plasmid, and 24 hr posttransfection cells were stimulated for 4 hr with 10 ng/ml of TNFα.
(A) KRC and KRCtr (B) dominant-negative and antisense KRC. Data from at least five experiments normalized for β-galactosidase activity are shown.
(C) Western blot analysis demonstrating that antisense KRC decreases the amount of endogenous KRC protein.
(D) Sense KRC inhibits while (E) antisense and dominant-negative KRC increases both the frequency of cells and the intensity of cytokine-driven NFκB transactivation per cell. (D) KRC, KRCtr, and (E) antisense KRC in the presence (right) and absence (left) of TNFα. 293T cells were cotransfected as above with the NFκB/GFP construct and the indicated plasmids. A CMV/GFP construct was used to monitor transfection efficiency, which was from 50% to 60% (data not shown).
Molecular Cell 2002 9, DOI: ( /S (01) )

4. Figure 3 Interaction of KRCtr with TRAF Family Members
(A) Schema of KRC constructs used.
(B) Left: Interaction of KRCtr with TRAF1 and TRAF2 in mammalian cells. 293T cells were cotransfected with the indicated FLAG-TRAFs and MYC-tagged KRCtr and were immunoprecipitated with anti-MYC antibody, followed by blotting with anti-FLAG antibody. Right: Direct Western blot of KRC full-length and KRCtr with anti-Xpress or anti-MYC.
(C) KRCtr interacts with TRAF2 lacking the RING finger domain. 293T cells were transfected with MYC-KRCtr and with FLAG-tagged TRAF2 or with TRAF2 DN (87–501).
(D) Interaction of KRCtr and full-length with endogenous TRAF2. 293T were transfected with an expression vector encoding a MYC-tagged KRCtr, Xpress-tagged KRC full-length, or empty plasmid. Lysates from 293T cells were incubated with anti-MYC or anti-Xpress antibodies. Coimmunoprecipitated endogenous TRAF2 was detected by Western blotting with specific anti-TRAF2 antibody.
(E) Interaction of endogenous KRC and TRAF2. Lysates from 293T cells were precleared with rabbit immunoglobulins and protein AG-sepharose, and the supernatants were incubated with KRC antisera or control rabbit antisera overnight. Immunoprecipitates were washed four times with 1 ml of lysis buffer, and Western blotting was performed with anti-TRAF2 antibody. Protein G-HRP was used to visualize TRAF2.
Molecular Cell 2002 9, DOI: ( /S (01) )

5. Figure 4 KRC Can Function Both in the Cytosol and the Nucleus
(A) Western blot analysis of endogenous KRC protein in nucleus (N) and cytosol (C) of 293T cells using a polyclonal anti-KRC antisera directed against the C terminus. Assessment of fraction integrity was performed using antibodies against hsp90 and Oct1.
(B) Fluorescence microscopy of endogenous KRC protein in 293T cell line using anti-KRC antisera.
(C) KRC prevents while antisense KRC increases TRAF2-dependent NFκB activation. Alteration of TRAF2-mediated activation of NFκB by ectopically expressed KRC or antisense KRC as demonstrated by transient transfection assays.
(D) FACS analysis of NFκB/GFP expression with sense or antisense KRC transfection 293T cells (3 × 105) were transfected with 25 ng of NFκB luciferase or GFP reporter plasmid, 50 ng of CMVβ-gal, and 1 μg of each indicated plasmid, and 24 hr posttransfection cells were harvested. Data from at least five experiments normalized for β-galactosidase activity are shown. Vec refers to the empty MYC vector without the addition of TRAFs.
Molecular Cell 2002 9, DOI: ( /S (01) )

6. Figure 5 KRC Increases TNFα-Induced Apoptosis
3T3 cells were cotransfected with CMV lacZ vector (300 ng per plate) and either empty expression vector or the expression vectors indicated (2 μg of each). Half of the transfected cultured cells were treated with TNFα (20 ng/ml) at 12 hr after the transfection, and the other half was left untreated. All the cells were fixed and stained at 36 hr after the transfection. The number of blue cells in each transfection was determined by counting six different fields. A representative experiment of three performed is presented.
Molecular Cell 2002 9, DOI: ( /S (01) )

7. Figure 6 KRC Prevents TRAF2- and TNFα-Dependent JNK Activation
Inhibition of TRAF2- (A) and TNFα- (B and C) mediated JNK/SAPK activation by ectopic expression of KRC.
(A) 293T cells were transfected with 400 ng of TRAF2 and 2 μg of the indicated expression vector. Twenty-four hours after the transfection, the cells were harvested and lysed, and the endogenous JNK was precipitated with 5 μg of GST-cJUN (1–79) for 4 hr. JNK activity was determined by using GST-cJUN (1–79) as a substrate.
(B and C) 293T cells were cotransfected with vectors encoding HA-tagged JNK2 (500 ng) and the indicated expression vector (2 μg). Twenty-four hours after the transfection, the cells were stimulated for 10 min with 10 ng/ml of TNFα and were harvested at varying time points. JNK activity was assayed with GST-cJUN (1–79) as substrate.
Molecular Cell 2002 9, DOI: ( /S (01) )

8. Figure 7
The Interaction of KRC with TRAF2 Is the Primary Means by which KRC Accomplishes Its Function
(A) FACS analysis of GFP-positive cells from 293T cells transfected with an NFκB/GFP reporter, antisense KRC, and various dominant-negative (DN) constructs including TRAF2DN, IKKβDN (KR), superinhibitor of NFκB (IKBα mutant), and NIK DN (AA).
(B) Coimmunoprecipitation of TNFR1 and TRAF2 in the presence or absence of antisense KRC in HeLa cells.
Molecular Cell 2002 9, DOI: ( /S (01) )