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Blood, Vol. 110, Issue 3, 962-971, August 1, 2007
Lysosome-associated small Rab GTPase Rab7b negatively regulates TLR4 signaling in macrophages by promoting lysosomal degradation of TLR4
Blood Wang et al.
110: 962
Supplemental materials for Wang et al, Vol. 110, Issue 3, 962-971
Files in this Data Supplement:
- Figure S1. Rab7b expression was regulated by LPS (JPG, 36.2 KB)
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Peritoneal macrophages (A) and Raw 264.7 cells (B) were treated with 100 ng/mL LPS as indicated. Expression level of Rab7b was examined by Western blotting using Rab7b polyclonal antibody.41  -actin was used as quantitative control.
- Figure S2. Rab7b is a small GTPase (JPG, 76.4 KB)
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(A) In vitro GTP binding assay of recombinant Rab7b GST fusion proteins. Indicated GST fusion proteins and sepharose 4B beads were incubated with 32P-labeled GTP in the presence of excesses of indicated competitors. After extensive washing, the bound radioactivity was counted in a scintillation counter. (B) In vitro GTPase activity assay. Purified GST fusion proteins (0.5 µg) were incubated with 32P-labeled GTP for 1 hour at 37°C. The extent of GTP hydrolysis was assessed by thin layer chromatography (TLC). GST-Ras was used as a positive control. (C,D) In vivo GTP-binding status of His-tagged Rab7b (mutants) in Raw 264.7 cells treated with or without 100 ng/mL LPS. Raw 264.7 stable transfectants were metabolically labeled with 32P-H3PO4 for 4 hours. The cells cultured under normal conditions were stimulated with (D) or without (C) LPS, as indicated. Lysates were then immunoprecipitated with Ni-beads, and bound GTP and GDP were separated on TLC. Results in (A), (C) and (D) are presented as means plus and minus a SD of triplicate samples.
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