Journal: Scientific Reports

Article Title: The mammalian CTLH complex is an E3 ubiquitin ligase that targets its subunit muskelin for degradation

doi: 10.1038/s41598-019-46279-5

Figure Lengend Snippet: RanBPM and TWA1 are essential for complex stability. Whole cell extracts prepared from control shRNA and RanBPM shRNA HEK293 cells ( a ), or from control (labelled as C), TWA1, RMND5A, MAEA, ARMC8 and muskelin HEK293 CRISPR knockout cells ( b – f ) were analyzed by Western blot with antibodies to CTLH complex members, as indicated. Vinculin was used as a loading control. Quantifications are shown below each blot and protein levels are shown relative to control cells set to 1 and normalized to Vinculin levels. Data represent averages from three separate experiments, with error bars indicating SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Gels were transferred to polyvinylidene fluoride membranes, blocked in 5% milk and hybridized with the following antibodies: ARMC8 (E-1, sc-365307; Santa Cruz Biotechnology, Santa Cruz, CA, USA); FLAG (M2, F1804, Sigma-Aldrich, St. Louis, MO, USA); HA (HA-7, H3663 Sigma-Aldrich); MAEA (AF7288, R&D Systems, Minneapolis, MN, USA); Muskelin (C-12, sc-398956, Santa Cruz Biotechnology); RanBPM (5M, 71-001, Bioacademia, Japan); RMND5A (NBP1-92337, Novus Biologicals, Littleton, CO, USA); TWA1 (NBP1-32596, Novus Biologicals); Vinculin (E1E9V, Cell Signaling Technology, Danvers, MA, USA); WDR26 (ab85962, Abcam, Cambridge, UK); β-actin (A5441, Sigma-Aldrich) and His (MAB050, R&D Systems).

Techniques: Control, shRNA, CRISPR, Knock-Out, Western Blot