![Figure 1. Nucleolin binds activation-resistant Fas and shows altered expression in B-cell lymphomas. (A) Silver-stained gel separating primary NHL and BJAB samples subjected to Fas activation and IP with agonistic antibody CH-11 (BJAB and primary NHL CH-11). The remaining lysates were subjected to a second Fas IP (B-10) of any remaining activation-resistant Fas (BJAB and primary NHL B-10). Specific activation-resistant Fas bands were excised, digested with trypsin, and analyzed by nanoflow-LC-MS/MS fragmentation and collision-induced dissociation spectra profiling. A 100 kDa band of interest (asterisk) reveals the protein that is the focus of the current study. The protein sequence of nucleolin is shown with peptides (red text) from the 100 kDa band identified by nanoflow-LC-MS/MS spectra. Peptides map to the RNA binding domains in the C-terminal region of nucleolin. (B) Whole-cell extracts from BJAB, Raji, Daudi, BC-3, and a histocytic lymphoma (U937) cell line and two B-lymphocyte isolations from healthy donors were subjected to IP with B-10 anti-Fas agarose, which recognizes an epitope within the cytoplasmic region of Fas, and analyzed by IB for the presence of nucleolin with MS-3 anti-nucleolin antibody. Representative data from 3 different experiments are shown. Shown is an analysis of 2 primary lymphoma samples (diffuse large B-cell lymphoma [DLBCL] and mantle cell lymphoma [MCL]) demonstrating nucleolin-Fas complex from a screen of 4 DLBCL and 6 MCL primary samples. Extracts were immunoprecipitated with B-10 and analyzed by IB for the presence of nucleolin in precipitated complexes. BJAB cells and isotype IP were used as positive and negative controls, respectively. β-actin was used as a loading control. (C) Whole-cell lysates of 5 lymphomas cell lines (BJAB, Raji, Daudi, BC-3, and U937) and healthy donor B-lymphocyte populations were analyzed by IB for nucleolin expression. β-actin was used as a loading control (bottom panel). Whole-cell lysates of 4 primary hematologic cancer tissues multiple myeloma (MM), MCL, chronic lymphocytic leukemia (CLL) and DLBCL were subjected to IB analysis of nucleolin. β-actin was used as a loading control. Ratio of nucleolin to β-actin levels determined by densitometry is shown below each sample lane. Representative data from 3 different experiments are shown.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/121/23/10.1182_blood-2012-12-471094/4/4729f1.jpeg?Expires=1735573600&Signature=raNYE3To4pwseSXMhv2xWlIVtcSQsbGzgeDc3dWn4whwcci~dueRqDCF-e3qCZ05cjthvpNlmIZfIefypOb9xm7whdK0fM9cpWj6~FgA-mmUqkVDm-Pa2-CdaBNR9UmPAE~vm660cb~zX0qBMLvbmpwhkeM5NLr5Hj0fMfs6G3kxLcoBUO8OF2bvPOw2kbUysL0pgDgi7rYqeprbwYrVqMYymNu3ha~Kb9GJS5fYiNVWs-zmHXZUMe~4eWdxIQ~owjgTWLPbS9K3WxOElPVbMHk8zsd9ZQt3YVVMm3Akn8ME4gfNZ-KdjtVOkuYUHi4rnA4lXu~sl3NkP-ORGtGXmQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Nucleolin binds activation-resistant Fas and shows altered expression in B-cell lymphomas. (A) Silver-stained gel separating primary NHL and BJAB samples subjected to Fas activation and IP with agonistic antibody CH-11 (BJAB and primary NHL CH-11). The remaining lysates were subjected to a second Fas IP (B-10) of any remaining activation-resistant Fas (BJAB and primary NHL B-10). Specific activation-resistant Fas bands were excised, digested with trypsin, and analyzed by nanoflow-LC-MS/MS fragmentation and collision-induced dissociation spectra profiling. A 100 kDa band of interest (asterisk) reveals the protein that is the focus of the current study. The protein sequence of nucleolin is shown with peptides (red text) from the 100 kDa band identified by nanoflow-LC-MS/MS spectra. Peptides map to the RNA binding domains in the C-terminal region of nucleolin. (B) Whole-cell extracts from BJAB, Raji, Daudi, BC-3, and a histocytic lymphoma (U937) cell line and two B-lymphocyte isolations from healthy donors were subjected to IP with B-10 anti-Fas agarose, which recognizes an epitope within the cytoplasmic region of Fas, and analyzed by IB for the presence of nucleolin with MS-3 anti-nucleolin antibody. Representative data from 3 different experiments are shown. Shown is an analysis of 2 primary lymphoma samples (diffuse large B-cell lymphoma [DLBCL] and mantle cell lymphoma [MCL]) demonstrating nucleolin-Fas complex from a screen of 4 DLBCL and 6 MCL primary samples. Extracts were immunoprecipitated with B-10 and analyzed by IB for the presence of nucleolin in precipitated complexes. BJAB cells and isotype IP were used as positive and negative controls, respectively. β-actin was used as a loading control. (C) Whole-cell lysates of 5 lymphomas cell lines (BJAB, Raji, Daudi, BC-3, and U937) and healthy donor B-lymphocyte populations were analyzed by IB for nucleolin expression. β-actin was used as a loading control (bottom panel). Whole-cell lysates of 4 primary hematologic cancer tissues multiple myeloma (MM), MCL, chronic lymphocytic leukemia (CLL) and DLBCL were subjected to IB analysis of nucleolin. β-actin was used as a loading control. Ratio of nucleolin to β-actin levels determined by densitometry is shown below each sample lane. Representative data from 3 different experiments are shown.
