e1htt-25q-rfp  (Evident Corporation)

Journal: Cell & Bioscience

Article Title: Siah-1-interacting protein regulates mutated huntingtin protein aggregation in Huntington’s disease models

doi: 10.1186/s13578-022-00755-0

Figure Lengend Snippet: In the presence of SIP, the levels of N-terminal HTT decrease and mutant HTT aggregates decrease. a SIP decreases the expression of proteins encoded by exon 1 of wt HTT in the cellular HD model. HEK293T cells co-transfected with plasmids that are indicated in the figure: control pEntry and e1HTT-25Q-RFP; wt SIP and e1HTT-25Q-RFP. b SIP decreases the aggregation of proteins encoded by exon 1 mHTT in the cellular HD model. HEK293T cells co-transfected with plasmids that are indicated in the figure: control pEntry and e1HTT-72Q-RFP; wt SIP and e1HTT-72Q-RFP. The left parts of panels a and b were imaged under a fluorescent microscope. Images in the right parts of panels a and b were taken under transmitted light. Scale bar = 1 µM. The size and number of mHTT aggregates (e1HTT-72Q) are presented in panels b ’ and b ”, respectively. Red color signals of e1HTT-72Q were measured as the number of pixels per aggregate b ’ and show the average aggregate size measured for each of the analyzed images. c , d Western blot (WB) analysis of HEK293T cells that co-expressed e1HTT-25Q c or e1HTT-72Q d and wt SIP or control pEntry plasmids. c , d Graphs show densitometric analysis of western blotting bands for the indicated proteins using GAPDH abundance for normalization and presented as fold changes. M, protein marker in kilodaltons (kDa). The results are expressed as mean ± SEM. * p < 0.05, *** p < 0.001. The results were obtained from at least three independent HEK293T culture preparations

Article Snippet: The expression of e1HTT-72Q aggregates and e1HTT-25Q protein in HEK293T cells that were transiently co-transfected with e1HTT-72Q-RFP or e1HTT-25Q-RFP plasmids and wt SIP or its dimerization mutants in pCMV-pEntry plasmid or pCMV-pEntry as a control were imaged using an IX70 Microscope (Olympus).

Techniques: Mutagenesis, Expressing, Transfection, Microscopy, Western Blot, Marker

Journal: Cell & Bioscience

Article Title: Siah-1-interacting protein regulates mutated huntingtin protein aggregation in Huntington’s disease models

doi: 10.1186/s13578-022-00755-0

Figure Lengend Snippet: SIP increases autophagy in cells that express N-terminal mutant HTT but not in the presence of N-terminal wt HTT. a HEK293T cells were co-transfected with plasmids (control pEntry and e1HTT-25Q-RFP; wt SIP and e1HTT-25Q-RFP; control pEntry and e1HTT-72Q-RFP; wt SIP and e1HTT-72Q-RFP), pretreated with rapamycin and chloroquine, and imaged for autophagy using the Cyto-ID Autophagy detection kit. Signals from autophagosomes were detected in green. Signals that corresponded to the expression of proteins that were encoded by e1HTT-25Q-RFP and e1HTT-72Q-RFP were detected in red and yellow, respectively. Aggregates of e1HTT-72Q were imaged in yellow (white arrows) because of their high fluorescence intensity, which was detected in the FITC channel. Scale bar = 1 µM. b Area of autophagosomes in double-transfected HEK293T cells using the above plasmids. The area of autophagosomes was measured as the number of pixels per e1HTT expressed cell

Article Snippet: The expression of e1HTT-72Q aggregates and e1HTT-25Q protein in HEK293T cells that were transiently co-transfected with e1HTT-72Q-RFP or e1HTT-25Q-RFP plasmids and wt SIP or its dimerization mutants in pCMV-pEntry plasmid or pCMV-pEntry as a control were imaged using an IX70 Microscope (Olympus).

Techniques: Mutagenesis, Transfection, Expressing, Fluorescence

Journal: Cell & Bioscience

Article Title: Siah-1-interacting protein regulates mutated huntingtin protein aggregation in Huntington’s disease models

doi: 10.1186/s13578-022-00755-0

Figure Lengend Snippet: Ubiquitination of N-terminal wt HTT and mHTT increases in the presence of SIP. Immunoprecipitations (IP) of ubiquitinated exon 1 wt HTT a or mHTT b in the presence of SIP detected by Western blot (WB). a Immunoprecipitation was performed in extracts from cells that were co-transfected with both control pEntry and e1HTT-25Q-RFP plasmids or both wt SIP and e1HTT-25Q-RFP plasmids. b Immunoprecipitation was performed in extracts from cells that were co-transfected with both control pEntry and e1HTT-72Q-RFP plasmids or both wt SIP and e1HTT-72Q-RFP plasmids. In a and b , IP was performed using anti-HTT N-terminal antibody (HTT) and analyzed for the presence of exon 1 HTT ubiquitination (e1HTT-25Q-Ub and e1HTT-25Q-polyUb or e1HTT-72Q-Ub and e1HTT-72Q-polyUb, respectively) with the application of an antibody that recognized ubiquitin (Ub; clone P4D1). In b , control IP presents the co-precipitation of exon 1 mHTT (e1HTT-72Q) using anti-HTT N-terminal antibody. # In the control IP experiments in panel a , bands at 55 kDa that corresponded to exon 1 of wt HTT fused with RFP tag (encoded by e1HTT-25Q-RFP) and IgG heavy chain could not be separated in 8% polyacrylamide gels. Ab, corresponding antibody subjected to IP; pEntry, control pEntry plasmid; SIP wt, wildtype SIP; Total, total protein extract; ►, co-precipitating exon 1 of wt HTT or mHTT or its ubiquitinated forms; IgG, heavy chain of the antibody; M, protein marker in kilodaltons (kDa). As controls in a , total protein extracts were analyzed by WB for the electrophoretic mobility of exon 1 wt and mHTT detected by N-terminal HTT antibody. SIP was detected by anti-SIP antibody and the loading control GAPDH was detected by anti-GAPDH antibody. c Level of ubiquitination in total protein extracts from HEK293T cells that were treated with different concentrations of the proteasome inhibitor MG132. d Densitometry of exon 1 mHTT ubiquitination (e1HTT-72Q-Ubiquitin) bands detected in the presence of wt SIP and control pLenti plasmid was calibrated based on the intensity of immunoprecipitated exon 1 mHTT (e1HTT-72Q) and presented as fold changes. Note that despite equal amounts of GAPDH in the total cell extracts there is less mHTT and less mHTT binds to the resin in the IP in the presence of wt SIP compared to control conditions. Therefore, the ubiquitination results from IP were normalized to the amount of HTT that bound to the resin. The results are expressed as mean ± SEM. * p < 0.05, ** p < 0.01. ns, not significant. The results were obtained from at least three independent HEK293T culture preparations

Article Snippet: The expression of e1HTT-72Q aggregates and e1HTT-25Q protein in HEK293T cells that were transiently co-transfected with e1HTT-72Q-RFP or e1HTT-25Q-RFP plasmids and wt SIP or its dimerization mutants in pCMV-pEntry plasmid or pCMV-pEntry as a control were imaged using an IX70 Microscope (Olympus).

Techniques: Western Blot, Immunoprecipitation, Transfection, Plasmid Preparation, Marker

Journal: Cell & Bioscience

Article Title: Siah-1-interacting protein regulates mutated huntingtin protein aggregation in Huntington’s disease models

doi: 10.1186/s13578-022-00755-0

Figure Lengend Snippet: Mutations in the N-terminal domain of SIP decrease the ubiquitination of exon 1 mHTT. a Immunoprecipitation (IP) of ubiquitinated exon 1 mHTT in the presence of SIP and its dimerization mutants detected by Western blot (WB). Immunoprecipitation was performed in extracts from cells that were co-transfected with pEntry and e1HTT-72Q-RFP plasmids, wt SIP and e1HTT-72Q-RFP plasmids, SIP K21W and e1HTT-72Q-RFP plasmids, or SIP T30R_S33E and e1HTT-72Q-RFP plasmids. Immunoprecipitation was performed using anti-HTT N-terminal antibody (HTT) and analyzed for the presence of exon 1 mHTT ubiquitination (e1HTT-72Q-Ub or e1HTT-72Q-polyUb) with the application of an antibody that recognized ubiquitin (Ub; clone P4D1). The detection of co-precipitated exon 1 mHTT (e1HTT-72Q) using anti-HTT N-terminal antibody is shown in the control IP. Ab, corresponding antibody subjected to immunoprecipitation; pE, control pEntry plasmid; SIP wt, wildtype SIP; SIP K21W, mutant SIP K21W; SIP T30R_S33E, mutant SIP T30R_S33E; Total, total protein extract; ►, co-precipitating exon 1 mHTT or its ubiquitinated forms. IgG, heavy chain of antibody; M, protein marker in kilodaltons (kDa). As controls in panel a , total protein extracts were analyzed by WB for the electrophoretic mobility of e1HTT-72Q protein that was detected by N-terminal HTT antibody. SIP was detected by anti-SIP antibody. The loading control, GAPDH, was detected by anti-GAPDH antibody. b Densitometry of exon 1 mHTT ubiquitination bands in the presence of SIP and its dimerization mutants, detected by IP was calibrated based on the intensity of immunoprecipitated e1HTT-72Q in control IP and presented as fold changes. b Note that despite equal amounts of GAPDH in the total cell extracts there is less mHTT and less mHTT binds to the resin in the IP in the presence of wt SIP compared to control conditions and mHTT levels are elevated in the presence of SIP mutants. Therefore, the ubiquitination results from IP were normalized to the amount of mHTT that bound to the resin. The results are expressed as mean ± SEM. * p < 0.05, *** p < 0.001. ns, not significant. The results were obtained from five independent HEK293T culture preparations

Article Snippet: The expression of e1HTT-72Q aggregates and e1HTT-25Q protein in HEK293T cells that were transiently co-transfected with e1HTT-72Q-RFP or e1HTT-25Q-RFP plasmids and wt SIP or its dimerization mutants in pCMV-pEntry plasmid or pCMV-pEntry as a control were imaged using an IX70 Microscope (Olympus).

Techniques: Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Mutagenesis, Marker

Journal: Cell & Bioscience

Article Title: Siah-1-interacting protein regulates mutated huntingtin protein aggregation in Huntington’s disease models

doi: 10.1186/s13578-022-00755-0

Figure Lengend Snippet: Mutations in the N-terminal domain of SIP decrease its ability to protect mHTT against aggregation. a HEK293T cells were co-transfected with plasmids (control pEntry and e1HTT-72Q-RFP; wt SIP and e1HTT-72Q-RFP; SIP K21W and e1HTT-72Q-RFP; SIP T30R_S33E and e1HTT-72Q-RFP) and imaged under a fluorescent microscope (left part of panel a ). Images in the right part of the panel a were taken in transmitted light. The size and number of mHTT aggregates (e1HTT-72Q) are presented in panels b and c , respectively. b Red color signals of e1HTT-72Q were measured as the number of pixels per aggregate. Panel b shows the average aggregate size measured for each of the analyzed images. The results are expressed as mean ± SEM. Scale bar = 1 µM. ** p < 0.01; *** p < 0.001. ns, not significant. The results were obtained from three independent HEK293T culture preparations

Article Snippet: The expression of e1HTT-72Q aggregates and e1HTT-25Q protein in HEK293T cells that were transiently co-transfected with e1HTT-72Q-RFP or e1HTT-25Q-RFP plasmids and wt SIP or its dimerization mutants in pCMV-pEntry plasmid or pCMV-pEntry as a control were imaged using an IX70 Microscope (Olympus).

Techniques: Transfection, Microscopy