Journal: Molecular and cellular biology
Article Title: β-TrCP-Mediated Proteolysis of Mis18β Prevents Mislocalization of CENP-A and Chromosomal Instability.
doi: 10.1080/10985549.2024.2382445
Figure Lengend Snippet: Figure 1. Protein levels but not the transcript levels of Mis18b are increased in b-TrCP depleted cells. (A and B) Western blots (A) and bar graphs (B) showing the protein levels of b-TrCP, Mis18b and CENP-A in MDA-MB-231D b-TrCP cells untreated or treated with DOX for 48 h. The protein levels of Mis18b and CENP-A in B were calculated after normalization with GAPDH that was used as a loading control and expressed as fold increase relative to No DOX control. (C and D) Gel images from semi-quantitative RT-PCR (C) and bar charts from RT-qPCR (D) showing the mRNA levels of b-TrCP, OIP5 (Mis18b) and CENPA in MDA-MB-231D b-TrCP cells untreated or treated with DOX for 48 h. The quantification in D was after the normalization with GAPDH that was used as an internal control and expressed as log2- fold difference. (E and F) Ubiquitin pulldown assay (E) showing the polyubiquitination of Mis18b in MDA-MB-231D b-TrCP cells with or without DOX treatment and bar chart (F) depicting the polyubiquitinated Mis18b levels normalized against input Mis18b levels. (G and H) Protein stability assay using cycloheximide (CHX) depicting the stability of Mis18b in both DOX treated as well as untreated cells until 6 h and quantification plot (H) showing the percent Mis18b remaining in similar conditions. The half-life of Mis18b in both the conditions was calculated using GraphPad Prism. Error bars depict standard deviation (SD) from three biological repeats and the P-values were calculated using Student’s t test in B, D, F and H.
Article Snippet: The beads were then washed three times with TBS-T at room temperature and the beads bound to the protein of interest were boiled with 60 mL of 2� Laemmli buffer at 95 �C for 10 min. Western blotting was done to detect the polyubiquitinated Mis18b by probing the nitrocellulose membranes with anti-OIP5 antibody (12142-1-AP, Proteintech).
Techniques: Western Blot, Control, Quantitative RT-PCR, Ubiquitin Proteomics, Stability Assay, Standard Deviation
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