Journal: PLoS ONE

Article Title: Induction of interferon-β and interferon signaling by TRAIL and Smac mimetics via caspase-8 in breast cancer cells

doi: 10.1371/journal.pone.0248175

Figure Lengend Snippet: MCF-7 cells were treated with LCL161 (10 μM) and/or TRAIL (100 ng/mL) for indicated time periods and total cell lysates were analyzed by immunoblotting for cIAP1 (A) the NFKB2 gene product p100 and p52 (B) or phosphorylated and total STAT1 (D). Intensity of bands in B and D were quantified (C and E) and normalized to LCL161+TRAIL (C) or TRAIL (E) treatment for 48 h. Protein levels of cIAP1 (F), p100 and p52 (G), or phosphorylated and total STAT1 (H) were measured in CAMA-1 cells that had been treated with LCL161 and/or TRAIL for 8 h in the absence or presence of zVAD-FMK (20 μM). Figures are representative of three independent experiments. Data in C and E indicate individual data points with bars representing the mean and error bars representing SEM, n = 3. * denotes p<0.05, ** p<0.01, *** p<0.001 using ANOVA followed by Tukey’s honest significance test.

Article Snippet: This was followed by incubation with primary antibodies against phosphorylated STAT1 (1:400, #9167), total STAT1 (1:400, #9172), FADD (1:400, #27825), caspase-8 (1:300, #9746), and FLIP (1:400, #56343) all from Cell Signaling Technology; p100/p52 (1:300, ab131539) and IFNAR1 (1:400, ab124764) from Abcam; cIAP1 (1:200, AF8181, R&D Systems); actin (1:2,000, #0869100, MP Biomedicals); and RIP1 (1:500, #610458, BD Biosciences).

Techniques: Western Blot

Journal: Biochemical and biophysical research communications

Article Title: Negative Feedback Regulation of NF-κB-Inducing Kinase is Proteasome-Dependent But Does Not Require Cellular Inhibitors of Apoptosis

doi: 10.1016/j.bbrc.2014.05.122

Figure Lengend Snippet: cIAP1/2 are not required for proteasome-dependent NIK turnover in HeLa cells. (A) HeLa cells were treated for four hours with either 1 μM GT13072 or GT13199. Cells were lysed and immunoblotted for the indicated proteins. (B) HeLa cells were treated with LIGHT or GT13072 for the times indicated. Lysates were immunoblotted for the indicated proteins. (C) HeLa cells were stimulated with either LIGHT or GT13072 for four hours. Cells were then treated with 2.5 μg/ml cycloheximide (CHX) for up to one hour (V, vehicle control). Cells were lysed and immunoblotted for the indicated proteins. (D) HeLa cells or WT MEFs were treated as in (C) plus 10 μM MG132 (+) for one hour (C, cycloheximide; V, vehicle control). Lysates were immunoblotted for the indicated proteins. (E) In unstimulated cells scant NIK abundance is maintained by a complex consisting of TRAF2 (T2), TRAF3 (T3) and cIAP1/2. The cIAPs ubiquitylate newly synthesized NIK (black circles) leading to its rapid proteasomal degradation (left side). Following ligation of receptors that activate non-canonical NF-κB, the basal regulatory complex is disrupted and NIK protein increases to detectable levels. Accumulated NIK then associates with and activates IKKα leading to induction of non-canonical NF-κB (right side). Activated IKKα also feeds back to phosphorylate NIK (P) triggering its degradation by the proteasome. Unlike basal NIK turnover, this feedback degradation of active NIK does not require cIAP1/2.

Article Snippet: Antibodies and reagents Recombinant human LIGHT, anti-cIAP1, and pan-cIAP antibodies were from R&D Systems.

Techniques: Synthesized, Ligation