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1) Product Images from "RTL8 promotes nuclear localization of UBQLN2 to subnuclear compartments associated with protein quality control"
Article Title: RTL8 promotes nuclear localization of UBQLN2 to subnuclear compartments associated with protein quality control
Journal: Cellular and molecular life sciences : CMLS
Figure Legend Snippet: RTL8, a family of small UBQLN2 interacting proteins, promotes nuclear translocation of UBQLN2. (A) Alignment of the protein sequences for the human (hRTL8) and murine (mRTL8) RTL8 proteins, showing a high degree of sequence homology. Similar residues are boxed in yellow; the bold typeface indicates that these residues are found in the majority of RTL8 homologs. Identical residues are bolded in white font and boxed in red. (B) Immunoblots of in vitro pull-down of recombinant purified protein GST-UBQLN2 and His-mRTL8A. α-UBQLN2 and α-RTL8 antibodies were used for immunoblotting. UBQLN2-mRTL8A interaction was confirmed by detection of mRTL8A in the eluate (E), flow-through (FT), and washes (W1–3) as shown. (C) Immunoprecipitated FLAG-UBQLN2 pull down of RTL8 from brain lysates of FLAG-tagged UBQLN2 transgenic mice. (D) Representative images showing mRTL8A-dependent nuclear translocation of UBQLN2 and co-localization into subnuclear puncta. HEK293 cells were co-transfected with FLAG-UBQLN2 ( green ) and control HA-empty vector or HA-mRTL8A (left panel and right series, respectively). DAPI ( blue ) stained nuclei. White outlines in the merged image mark nuclei. (Scale bar = 20 μm). (E) Representative images showing mRTL8A-mediated nuclear translocation of endogenous (endog) UBQLN2. HEK293 cells were transfected with HA-mRTL8A and stained for endogenous UBQLN2 ( green ) and HA-mRTL8A ( red ). Merged images include DAPI to highlight nuclei ( blue ). Arrows highlight cells demonstrating localization of endogenous UBQLN2 to nuclear puncta containing HA-mRTL8A. Arrowheads identify cells not expressing HA-mRTL8A in which UBQLN2 is diffusely cytoplasmic (Scale bar = 10 μm). (F) Representative images showing fluorescence recovery after photobleaching (FRAP) of UBQLN2-mRTL8A puncta, demonstrating their dynamic nature. HEK293 cells were transfected with iRFP-UBQLN2 ( pink ), mFAP10-mRTL8A ( green ) and mApple ( red ). mApple serves as a morphology marker. (Scale bar = 5 μm). ( right ) iRFP fluorescence recovery following bleach was plotted as a percentage of the average fluorescence intensity of the puncta over time. Data are shown as mean ± SD. N=17 puncta.
Techniques Used: Translocation Assay, Sequencing, Western Blot, In Vitro, Recombinant, Purification, Immunoprecipitation, Transgenic Assay, Mouse Assay, Transfection, Plasmid Preparation, Staining, Expressing, Fluorescence, Marker
Figure Legend Snippet: UBQLN2’s UBA domain is dispensable for interaction with mRTL8A, but required for UBQLN2 incorporation into mRTL8A subnuclear puncta. (A) Schematic depicting wild-type (WT) UBQLN2, various domain deletion constructs lacking the UBL domain (ΔUBL), PXX repeat region (ΔPXX) or UBA domain (ΔUBA), and the ubiquitin binding-deficient L619A mutation (represented as a red circle). (B) Pull-down of recombinant His-mRTL8A by recombinant full-length GST-UBQLN2 full-length WT, ΔUBL, and ΔUBA proteins. The flow-through (FT) and eluate were visualized on an immunoblot with α-UBQLN2 ( top ) or α-RTL8 antibodies ( bottom ). (C) Representative images of transfected HEK293 cells expressing HA-mRTL8A (red) and either FLAG-UBQLN2 WT, ΔUBL, ΔPXX, ΔUBL or L619A (green).White outlines in zoomed images delimit the nucleus as determined by DAPI staining (blue in merged images). White arrows in cells expressing FLAG-UBQLN2 L619A and HA-mRTL8A indicate partial co-localization of UBQLN2 and mRTL8A in the nucleus (Scale bar for merged images = 10 μm and zoomed images= 5 μm). (D) Quantification of co-localization of WT or domain-deleted UBQLN2 constructs with mRTL8A. MOC was calculated for each cell expressing both constructs in a field of view. Outliers as determined by a Grubbs’ test were excluded, following which a one-way ANOVA (p
Techniques Used: Construct, Binding Assay, Mutagenesis, Recombinant, Transfection, Expressing, Staining