Fig. 1: BAP1 deubiquitinates and controls MYCN protein stability. | Cell Death & Disease

Fig. 1: BAP1 deubiquitinates and controls MYCN protein stability.

From: Deficiency of BAP1 inhibits neuroblastoma tumorigenesis through destabilization of MYCN

Fig. 1

A, B Immunoblot (IB) analyses of whole-cell lysate (WCL) from BE2C (A) and IMR32 (B) cells treated with different concentration of MG132 for 10 h. C In vivo ubiquitination analysis of WCL and His pulldown of 293T cells transfected with the indicated plasmids. Forty hours after transfection, cells were treated with 20 μM MG132 for 6 h before harvesting. Ni-NTA, nickel-nitrilotriacetic acid. DF IB analysis of WCL derived from 293T cells transfected with the indicated plasmids. G IB analysis of WCL derived from 293T cells transfected with HA-MYCN and Flag-BAP1 constructs. Cells were treated with 100 μg/ml cycloheximide (CHX) and collected at the indicated time points. H MYCN protein abundance in (G) was quantified by ImageJ and plotted as indicated (H). I Immunoblot analyses of WCL and immunoprecipitates from 293T cells transfected with plasmids expressing the indicated proteins. Twenty-four hours after transfection, cells were treated with MG132 (10 μM) for 10 h before they were harvested. His-Ub histidine-tagged ubiquitin. J IB analysis of WCL derived from 293T cells transfected with HA-MYCN and GST-BAP1-WT/C91S plasmids. K IB analysis of WCL derived from 293T cells transfected with HA-MYCN and GST-BAP1-WT/C91S constructs. Cells were treated with 100 μg/ml cycloheximide (CHX) and collected at the indicated time points. L MYCN protein abundance in (K) was quantified by ImageJ and plotted as indicated (L). M Immunoblot analyses of WCL and immunoprecipitates from 293T cells transfected with plasmids expressing the indicated proteins. Twenty-four hours after transfection, cells were treated with MG132 (10 μM) for 10 h before they were harvested. His-Ub, histidine-tagged ubiquitin. N BAP1-knockdown cells (shBAP1–3 and shBAP1-6) as well as control BE2C cells (shScr) were treated with 100 μg/ml cycloheximide (CHX) for the indicated time period before they were harvested. Equal amounts of WCL were immunoblotted with the indicated antibodies. O MYCN protein abundance in (N) was quantified by ImageJ and plotted as indicated (O). P In vivo ubiquitination analysis of WCL and His pulldown of 293T cells transfected with the His-Ub plasmids. Forty hours after transfection, cells were treated with 20 μM MG132 for 6 h before harvesting. Ni-NTA, nickel-nitrilotriacetic acid. Q, R Immunoblot (IB) analyses of whole-cell lysate (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with indicated constructs. Thirty hours post-transfection, cells were treated with 10 μM MG132 for 10 h before harvesting. S Representative images for immunohistochemical staining of BAP1 and MYCN in human NB tumors and adrenal gland. Scale bars, 50 μm.

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