Figure 6(A) Subcellular distribution of HuR and Ago2 in Huh7 cells. Isotonic lysates of Huh7 cells transfected with HA-HuR expression or pCIneo control vector were analyzed on 3-30% OptiprepTM gradients for separation of organelles and localization of Ago2 and HuR were determined by western blotting analysis. Alix and Calnexin were used as markers of late endosome/MVB and ER respectively. Positions of specific organelle containing fractions were marked above the panels. * denotes the position of the high molecular weight HuR bands detected in the MVB fractions.(B) Detection of ubiquitinated HuR in Huh7 cells expressing HA-Ub. HuR was immunoprecipitated with anti-HuR antibody from cell lysate of Huh7 cells transfected with HA-Ub expression or pCIneo (control) plasmids and western blotted for HA or HuR (left panel). In vitro ubiquitination assay was done with recombinant His-tagged HuR and cell extract of Huh7 cells expressing HA-Ub. The scheme of the experiment and western blot data for input and cell extract along with immunoprecipitated materials are shown. Specific bands of Ub-HuR are marked by arrows and HuR is marked by arrowhead.(C) Changes in relative level of miR-122 in the subcellular ER and MVB associated fractions of OptiprepTM gradients done with the cell lysates from pCIneo or HA-HuR expression vector transfected cells were measured by qRT-PCR (mean+/- s.e.m., n=5).(D) Detection of ubiquitinated HuR in HuR-immunoprecipitated materials obtained from ER and MVB enriched fractions of Huh7 cells. Organelles were separated on OptiprepTM gradients, and HuR in organeller lysates were immunoprecipitated with HuR specific antibody and western blotted for ubiquitin to detect ubiquitinated form of HuR. Whole cell lysates used for optiprep analysis were western blotted for ubiquitinated proteins. Positions of the high molecular weight bands of ubiquitinated HuR are marked by arrowheads. Levels of HuR in the immunoprecipitated materials were detected using anti-HuR antibody. Anti-GFP antibody immnoprecipitated materials were used as control.(E) Detection of ubiquitinated HuR in HuR-immunoprecipitated materials obtained from ER and MVB enriched fractions of cells co-expressing HA-Ubiquitin and FLAG-HuR. Cells were lysed and organelles were separated on OptiprepTM gradients, and HuR in organeller lysates were immunoprecipitated with HuR specific antibody and western blotted for HA to detect ubiquitinated form of HuR. HuR in the lysate of the FLAG-HuR and HA-Ub co expressing cells were detected by HuR specific antibody.(F) Loss of miRNA association of HuR on MVB. HA-HuR expressing Huh7 cell lysates separated on OptiprepTM gradients were used for immunoprecipitation of HA-HuR and associated miRNA levels were measured by qRT-PCR and normalized against immunoprecipitated proteins. Levels of immunoprecipitated HA-HuR were detected by western blotting and quantified by densitometry. Data represented as mean+/- s.e.m., n=3.(G) Loss of miRNA from ubiquitinated form of HuR. Scheme of the double immunoprecipitation experiment is shown in the left panel. Western blot analysis for HuR in the anti-HA and anti-HuRimmunoprecipitatedFLAG-HuR along with the input of FLAG-HuR used. Lysates of cell expressing the FLAG-HuR and used for FLAGHuR purification is also shown (right upper panels). qRT-PCR based estimation of the miRNA per unit of HuRimmunoprecipitated were estimated for both HA and HuRimmunoprecipitated materials and normalized against respective protein levels quantified by densitometric quantification of western blots (right lower panel). Data represented as mean+/- s.e.m., n=3. Position of ubiquitinated band of FLAG-HuR is marked by arrow.(H) Increase in ubiquitinated form of HuR in Starved Huh7 cells. Levels of ubiquitinated HuR in Fed and Starved (5h) HA-Ub expressing Huh7 cells treated either with ethanol or MG132 (20µM) for same duration. The lysates were immunoprecipitated with HuR and western blotted for HA and HuR. Position of ubiquitinated bands were marked by arrows. The levels of ubiquitinated HuR were measured by densitometry and relative increase in ubiquitinated HuR levels against the non-ubiquitinated form were measured and plotted (mean+/- s.e.m., n=3).(I) Full length and a truncated version of HA-HuR without the hinge region (graphical scheme in upper panel) was used to score the effect of deletion of hinge region on EV associated miR-122 content measured by qRT-PCR (bottom panel). Western blots to check expression of HuR and its truncated variants are also shown." * " denotes the HA-HuR band (left lower panel). Data represented as mean+/- s.e.m., n=3.(J) Ubiquitination status of full length and a truncated version of HA-HuR without the hinge region between RRMII and RRMIII in Huh7 cells. HuR (full lenght or truncated version) were immunoprecipitated from cell lysates of Huh7 cells expressing these proteins and the immunoprecipitated materials were western blotted for HA-HuR and ubiquitination. Ubiquinitated HuR bands are marked by arrows. postion of non-ubiquitinated proteins were marked by arrowheads (upper panel). qRT-PCR based estimation of associated miRNA per unit of HA-HuR immunoprecipitated materials were estimated and normalized against respective protein levels quantified by densitometric quantification of western blots (lower panel). Data represented as mean+/- s.e.m., n=3. PCIneo transfected cells were used as control in immunoprecipitation reaction but no amplification of miR-122 were detected for HA immunoprecipitated materials from pCIneo tranfected cells.