![Fig. 1-1. RANK expression and activation of MAPK pathways in the HD-LM2 cell line. / (A) RANK expression in 4 HD cell lines as determined by Western blot (anti-RANK antibody used was from Santa Cruz, CA; SC9072). For the activation studies, HD-LM2 cell line cells (2 x 106/mL) were treated with 10 nM RANKL for 0 to 60 minutes. (B) Fifty micrograms of the protein extracts was probed with phospho-ERK (p44/42) antibodies (Santa Cruz, CA) (upper panel). Thirty micrograms of the same protein extracts was probed with ERK1 antibodies (lower panel). (C) Fifty micrograms of the protein extracts was resolved on the gels and probed with phospho-p38 MAPK antibodies (New England BioLabs, Beverly, MA) (upper panel). The same blot was stripped and reprobed with antibodies against the p38 MAPK (lower panel). (D) One hundred micrograms of whole cell protein was reacted with JNK1 antibodies (Santa Cruz, CA) and then immunoprecipitated with protein A/G sepharose. The beads were washed and subjected to kinase assay (upper panel). The c-Jun kinase assay was performed by a modified method as described,1-4 using washed beads as a source of enzyme and glutathione S-transferase-Jun (1-79) as substrate (2 μg/sample) in the presence of 10 μCi (.37 MBq) [32P] adenosine 5′-triphosphate (ATP) per sample. The kinase reaction was carried out by incubating the above mixture at 30°C in kinase assay buffer for 15 minutes. The reaction was stopped by boiling beads in sodium dodecyl sulfate (SDS) sample buffer. Finally, protein was resolved on 10% SDS-polyacrylamide gel electrophoresis (PAGE) gel. The radioactive bands of the dried gel were visualized and quantitated by phosphorImager. Forty micrograms of these same protein extracts was probed with JNK1 antibodies (lower panel). (E) HD-LM2 cells were treated with increasing concentrations of RANKL (0-10 nM) for 20 minutes. One hundred micrograms of whole cell protein was incubated with JNK1 antibodies and then immunoprecipitated with protein A/G sepharose. The beads were washed and subjected to kinase assay (upper panel). Forty micrograms of the same protein extracts was probed with JNK1 antibodies (lower panel).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/99/9/10.1182_blood-2002-01-0122/6/h80922588001.jpeg?Expires=1724232719&Signature=HR9fzp0uKmZZQr8exCro3EqeLhGAHwKGuK6qrwvVoO3sJ1-MqjZjQ4ehB~wCiXpf0U8xucoiY4JGEXnwyhszxD-Zjd4iQ54gpq1h5E7Gkxk5AyLsijsuOrVFcKf6XsoLvfXPJZfTTuvj69itCmGvD8xnMW0aq3CsQRLhgwONT7p9l3e~6Y-8q0eiDbp3y6hjZKuEPtZsM9IHX-FpaZH5yEWNCvrwk0XLnQkC0M2ieTkjW08hL2kG3bXTI2P43g3tNXzq9mnpJCrlYF5ZjLvX4LMIf06aYOUhY1OarULng~WVSohqVONXtPqIHcMcld1ZVkcNIU44KQ6vioIQD8tm4g__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
RANK expression and activation of MAPK pathways in the HD-LM2 cell line.
(A) RANK expression in 4 HD cell lines as determined by Western blot (anti-RANK antibody used was from Santa Cruz, CA; SC9072). For the activation studies, HD-LM2 cell line cells (2 x 106/mL) were treated with 10 nM RANKL for 0 to 60 minutes. (B) Fifty micrograms of the protein extracts was probed with phospho-ERK (p44/42) antibodies (Santa Cruz, CA) (upper panel). Thirty micrograms of the same protein extracts was probed with ERK1 antibodies (lower panel). (C) Fifty micrograms of the protein extracts was resolved on the gels and probed with phospho-p38 MAPK antibodies (New England BioLabs, Beverly, MA) (upper panel). The same blot was stripped and reprobed with antibodies against the p38 MAPK (lower panel). (D) One hundred micrograms of whole cell protein was reacted with JNK1 antibodies (Santa Cruz, CA) and then immunoprecipitated with protein A/G sepharose. The beads were washed and subjected to kinase assay (upper panel). The c-Jun kinase assay was performed by a modified method as described,1-4 using washed beads as a source of enzyme and glutathione S-transferase-Jun (1-79) as substrate (2 μg/sample) in the presence of 10 μCi (.37 MBq) [32P] adenosine 5′-triphosphate (ATP) per sample. The kinase reaction was carried out by incubating the above mixture at 30°C in kinase assay buffer for 15 minutes. The reaction was stopped by boiling beads in sodium dodecyl sulfate (SDS) sample buffer. Finally, protein was resolved on 10% SDS-polyacrylamide gel electrophoresis (PAGE) gel. The radioactive bands of the dried gel were visualized and quantitated by phosphorImager. Forty micrograms of these same protein extracts was probed with JNK1 antibodies (lower panel). (E) HD-LM2 cells were treated with increasing concentrations of RANKL (0-10 nM) for 20 minutes. One hundred micrograms of whole cell protein was incubated with JNK1 antibodies and then immunoprecipitated with protein A/G sepharose. The beads were washed and subjected to kinase assay (upper panel). Forty micrograms of the same protein extracts was probed with JNK1 antibodies (lower panel).
