Journal: Journal of Molecular Cell Biology

Article Title: Stability and function of RCL1 are dependent on the interaction with BMS1

doi: 10.1093/jmcb/mjad046

Figure Lengend Snippet: RCL1 is degraded through the ubiquitination–proteasome pathway. ( A and B ) After BMS1 knockdown by sh08, Hep3B ( A ) or HUH7 ( B ) cells were treated with leupeptin, aprotinin, MG132, pepstatin A, bortezomib, tripterin, or 3-MA, followed by western blot analysis to examine protein levels of BMS1 and RCL1. DMSO: negative control. ( C ) bms1l sq163 /sq163 mutant zebrafish were treated with MG132, bortezomib, pepstatin A, or leupeptin from 2 dpf to 5 dpf, followed by western blot analysis to examine protein levels of Bms1l and Rcl1 at 5 dpf. ( D ) Co-IP analysis of interactions between HA-ubiquitin and zebrafish Flag-Rcl1 (Z-Flag-Rcl1) or human FLAG-RCL1 (H-FLAG-RCL1). ( E ) Co-IP analysis of interactions among HA-ubiquitin, H-FLAG-RCL1, and BMS1. The transfected 293T cells were treated with MG132 for 24 h before cell harvest. ( F and G ) WISH analysis using liver-specific fabp10a probe in bms1l sq163 /sq163 mutant and siblings after MG132 or bortezomib treatment at 5 dpf. The area ratio of liver to eye was calculated. *** P < 0.001.

Article Snippet: The inhibitors and their final concentrations were as follows: leupeptin (MCE, HY-18234A) at 30 μM, aprotinin (MCE, HY-P0017) at 6 μM, MG132 (MCE, HY-13259) at 14 μM, pepstatin A (MCE, HY-P0018B) at 2 μM, bortezomib (MCE, HY-10227) at 60 nM, tripterin (MCE, HY-13067) at 2.5 μM, and 3-MA (MCE, HY-19312) at 5 mM.

Techniques: Ubiquitin Proteomics, Knockdown, Western Blot, Negative Control, Mutagenesis, Co-Immunoprecipitation Assay, Transfection

Journal: Cells

Article Title: WWOX Induction Promotes Bcl-X L and Mcl-1 Degradation Through a Lysosomal Pathway upon Stress Responses

doi: 10.3390/cells15030270

Figure Lengend Snippet: WWOX promotes Bcl-X L and Mcl-1 protein downregulation through a lysosomal degradation pathway. ( A ) Wwox +/+ MEFs were treated with serum-free medium for 36 h with or without the presence of a proteasome inhibitor, MG132 (1 μM), during the last 18 h of serum starvation. ( B ) Wwox +/+ MEFs were treated with serum-free medium for 32 h with or without the presence of NH 4 Cl (25 mM) or a lysosome inhibitor, chloroquine (CQ; 100 μM), during the last 4 and 8 h of serum starvation. ( C ) Wwox +/+ MEFs were treated with serum-free medium for 32 h with or without the presence of a lysosomal enzyme inhibitor E64d (30 μM) or pepstatin A (15 μM) during the last 4 and 8 h of serum starvation. Bcl-X L , Mcl-1, and WWOX protein expression was examined by Western blotting. β-actin was used as a loading control. Inhibition of Itch protein degradation in cycloheximide (CHX)-treated cells was used as a MG132 reagent control. The representative results from four repeated experiments are shown. Densitometric analysis for quantification of protein levels is shown in .

Article Snippet: Pepstatin A was obtained from Tocris (Bristol, UK).

Techniques: Expressing, Western Blot, Control, Inhibition