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(A) Wild-type or TRAF6-deficient bone marrow macrophages (BMM) retrovirally-rescued with wild-type (WT), RING mutant (C70A), or lysine-deficient (ΔK) full-length versions of FLAG-TRAF6 were treated as indicated with RANKL, then lysed and subjected to immunoblotting against the activated phosphorylated forms of IκBα, JNK, and p38. B , BMM described in (A) were replated and cultured with M-CSF and RANKL for 5 days to induce osteoclast differentiation. (C) Osteoclasts depicted in (B) were fixed and subjected to <t>TRAP</t> <t>solution</t> assay and quantified at 405 nm absorbance. (D) Total cell counts per well of retrovirally-rescued osteoclasts depicted in (B) as defined by cells containing at least 3 nuclei and being at least 100 µM in diameter.
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(A) Wild-type or TRAF6-deficient bone marrow macrophages (BMM) retrovirally-rescued with wild-type (WT), RING mutant (C70A), or lysine-deficient (ΔK) full-length versions of FLAG-TRAF6 were treated as indicated with RANKL, then lysed and subjected to immunoblotting against the activated phosphorylated forms of IκBα, JNK, and p38. B , BMM described in (A) were replated and cultured with M-CSF and RANKL for 5 days to induce osteoclast differentiation. (C) Osteoclasts depicted in (B) were fixed and subjected to TRAP solution assay and quantified at 405 nm absorbance. (D) Total cell counts per well of retrovirally-rescued osteoclasts depicted in (B) as defined by cells containing at least 3 nuclei and being at least 100 µM in diameter.

Journal: PLoS ONE

Article Title: TRAF6 Autoubiquitination-Independent Activation of the NFκB and MAPK Pathways in Response to IL-1 and RANKL

doi: 10.1371/journal.pone.0004064

Figure Lengend Snippet: (A) Wild-type or TRAF6-deficient bone marrow macrophages (BMM) retrovirally-rescued with wild-type (WT), RING mutant (C70A), or lysine-deficient (ΔK) full-length versions of FLAG-TRAF6 were treated as indicated with RANKL, then lysed and subjected to immunoblotting against the activated phosphorylated forms of IκBα, JNK, and p38. B , BMM described in (A) were replated and cultured with M-CSF and RANKL for 5 days to induce osteoclast differentiation. (C) Osteoclasts depicted in (B) were fixed and subjected to TRAP solution assay and quantified at 405 nm absorbance. (D) Total cell counts per well of retrovirally-rescued osteoclasts depicted in (B) as defined by cells containing at least 3 nuclei and being at least 100 µM in diameter.

Article Snippet: TRAP solution substrate and Coumermycin A were purchased from Sigma (St. Louis, MO).

Techniques: Mutagenesis, Western Blot, Cell Culture