Figure 2
RLIM p.(Tyr421Cys) is poorly expressed and readily degraded by the proteasome. (a) Human RLIM wild-type (WT) or Y421C TOKAS variant were expressed at increasing amounts (50–400 ng plasmid DNA) in Rlim-/y mESCs. RLIM expression was determined by immunoblotting and ERK1/2 expression analysed as a loading control. (b) Quantification of human RLIM wild-type (WT) or Y421C TOKAS variant protein expression from 50 ng plasmid cDNA in Rlim-/y mESCs. RLIM expression was determined by immunoblotting. Normalised data are represented as mean ± standard error of the mean (n = 3). Statistical significance was determined by student’s t-test. Figure was generated using BioRad ImageLab v6.0.1 (https://www.bio-rad.com/en-uk/product/image-lab-software?ID=KRE6P5E8Z) and Graphpad Prism v8.0 (https://www.graphpad.com/scientific-software/prism/). (c) Human RLIM wild-type (WT), Y421C TOKAS variant or W600Y catalytically-inactive variant were expressed in Rlim-/y mESCs. (Top panel) Human RLIM protein expression was determined by immunoblotting and ERK1/2 expression analysed as a loading control. (Bottom panel) Human RLIM mRNA expression was determined by qRT-PCR and normalised to Gapdh mRNA expression. Data are represented as mean ± standard error of the mean (n = 3). Statistical significance was determined by one-way ANOVA. Figure was generated using BioRad ImageLab v6.0.1 (https://www.bio-rad.com/en-uk/product/image-lab-software?ID=KRE6P5E8Z) and Graphpad Prism v8.0 (https://www.graphpad.com/scientific-software/prism/). (d) Human RLIM wild-type (WT) and Y421C TOKAS variant were expressed in Rlim-/y mESCs. RLIM localisation was determined by immunofluorescence. Scale Bar = 20 μm. Microscopy images were acquired using Zeiss ZEN 2012 SP5 (black edition) software (https://www.zeiss.com/microscopy/us/products/microscope-software/zen.html) and processed using NIH Image J v1.48 (https://imagej.net/) and Adobe Photoshop CC v19.1.8 (https://www.adobe.com/uk/products/photoshop.html). (e) Human RLIM wild-type (WT) or Y421C TOKAS variant were expressed in Rlim-/y mESCs treated with vehicle or 10 μM MG132 for 4 h. RLIM expression was determined by immunoblotting and ERK1/2 expression analysed as a loading control.
