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Blood, Vol. 108, Issue 4, 1320-1327, August 15, 2006
Abnormal disulfide-linked oligomerization results in ER retention and altered signaling by TNFR1 mutants in TNFR1-associated periodic fever syndrome (TRAPS)
Blood Lobito et al.
108: 1320
Supplemental material for: Lobito et al
Files in this Data Supplement:
- Figure S1. Mutant TNFR1 co-immunoprecipitates with wild-type receptors only in disulfide-linked oligomers (PDF, 133 KB) -
For immunoprecipitations 293T cells were transiently transfected with the indicated constructs. Anti-HA agarose beads were added to the lysates and this was rotated overnight at 4°C. The beads were then washed and heated to 80°C in LDS sample loading buffer with (A) or without (B) 6.25 mM DTT and run on SDS-PAGE. Immunoblottes were performed with the indicated antibodies. (B) Asterisk indicates a non-specific band and arrowhead indicates predicted size of TNFR1- CD-FLAG monomer.
- Figure S2. Additional data on mutant TNFR1 trafficking (PDF, 25.2 KB) -
(A) N-terminal FLAG-tagged CD TNFR1-YFP were transfected into 293T cells and surface (black line) or intracellularly (gray line) stained with anti-TNFR1 and analyzed by FACS. The histograms are gated on the YFP+ cells that were live according to forward and side scatter characteristics. The filled gray and black areas represent cells transfected with an YFP expression vector and stained as above. (B) TNFR1 internalization assay. 293T cells were transfected with the indicated TNFR1 constructs (CD, N-terminal FLAG-tag, C-terminal GFP fusion), and stained with anti-FLAG for the indicated periods of time. The cells were then acid stripped, washed and permeabilized, and stained with anti-mouse-PE. The percentage of GFP positive cells staining with anti-FLAG before (surface) or after (internal) permeablization is shown.
- Figure S3. Mutant TNFR1 does not traffic to the cells surface at lower protein expression levels (PDF, 69 KB) -
(A) HT1080 cells were transfected as with 0.12 µg WT FLAG-TNFR1, the indicated 0.05 µg HA-TNFR1-YFP and 0.5 µg pcDNA3 as carrier DNA. Surface TNFR1 expression was determined using anti-HA and FACS analysis. Insets are the geometric mean for anti-HA fluorescence of the YFP+ gated population. (B) TNFR1 expression levels in double transfected HT1080. Lysates from the transfected HT1080 cells were run on SDS-PAGE and immunoblotted with anti-TNFR1. The relative expression levels of HA-TNFR1-YFP and FLAG-TNFR1 were determined with densitometry. (C) Surface expression of H22Y-TNFR1-YFP in a stable HT1080 cell line. HT1080 cells were transfected as indicated and selected with G418 containing medium. Protein expression was determined by anti-TNFR1 immunoblotting cell lysates and anti-HA was used to determine surface expression as above.
- Figure S4. TRAPS mutant TNFR1 fail to induce ER stress responses (PDF, 72.5 KB) -
(A,B) Examination of UPR induction by mutant TNFR1 by BiP induction. (A) HT1080 cells were transfected with the indicated constructs. As indicated, some cells were treated with 2 µM Thapsigargin for 16 hr. 48 hr after transfection cells were lysed and western blots were performed on cell lysates using anti-BiP and anti-actin antibodies. The histogram shows BiP band intensity normalized against actin as a protein loading control. (B) HT1080 cells were transfected with the indicated constructs along with a BiP promoter-Luciferase reporter construct (kind gift of Amy Lee, University of Southern California) and Renilla luciferase as a transfection control. 48 hr later cells were lysed and luciferase activity was measured as described. Results are shown with BiP induction normalized against the YFP transfected samples. (C) Examination of EOR induction by mutant TNFR1. HT1080 cells were transfected with CD TNFR1 constructs at various concentrations with a NF- B luciferase reporter and Renilla luciferase as a transfection control. After 48 hr luciferase activity was examined as above.
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