human rtp801  (Databank Inc)

Journal: Oncotarget

Article Title: Loss of NEDD4 contributes to RTP801 elevation and neuron toxicity: implications for Parkinson's disease

doi: 10.18632/oncotarget.11020

Figure Lengend Snippet: a. R TP801 can be degraded by both the ubiquitin proteasome system and by the lysosomal pathway. NGF-differentiated PC12 cells or cortical neurons were treated during 4 hours with 1 μM epoxomycin, 10 μM MG132 or 50 μM chloroquine, and cell lysates were subjected to Western Blot. Membranes were probed first for RTP801 and then with α-actin, as a loading control. All samples were immunoblotted in the same membrane, but one irrelevant lane was deleted in the second panel. Graphs represent densitometric values (mean ± SEM) normalized to α-actin of three independent experiments in triplicates. Student's t -test, *** P < 0.001 and * P < 0.05 versus controls. b. NEDD4 polyubiquitinates RTP801 in a cell free assay. Recombinant NEDD4 E3 ligase, recombinant GST-RTP801, UbcH5b E2 enzyme, E1 enzyme, biotinylated ubiquitin and ATP were mixed and incubated at 37°C for 90 min. RTP801 was immunoprecipitated and immunocomplexes were analyzed by Western Blot. The membrane was incubated with Avidin/Biotin and then with chemiluminiscent peroxidase substrate solution (upper panel) and reprobed for RTP801 (lower panel). A representative image of three independent experiments is shown. (HMW Ub-RTP801, high molecular weight ubiquitinated RTP801) c. . HEK293 cells were transfected with pHAGE or pHAGE-NEDD4 along with HA-ubiquitin and pCMS-eGFP-RTP801 constructs. Forty-eight hours post-transfection either RTP801 was immunoprecipitated or non-specific rabbit immunoglobulins (Rb IgG) were added. Whole cell lysates (inputs) and the immunocomplexes were analyzed by Western Blot with anti-HA, anti-RTP801, anti-NEDD4 and anti-GAPDH (loading control) antibodies. A representative image of two independent experiments is shown. HMW Ub-RTP801 = High molecular weight ubiquitinated RTP801; IP = immunoprecipitation; IB = immunoblot. d. NEDD4 polyubiquitinates RTP801 with Ub-K63 chains. HEK293 cells were transfected with pCMS-eGFP-RTP801, along with pRK5-HA-Ub-K48 or pRK5-HA-Ub-K63 and pHAGE or pHAGE-NEDD4 as indicated. Forty-eight hours later, cultures were harvested and RTP801 was immunoprecipitated. Non-specific rabbit immunoglobulins (Rb IgG) were used as a negative control. Whole cell lysates (inputs) and RTP801 immunocomplexes were resolved in a Western Blot. Membrane was probed for HA, and reprobed for RTP801, for NEDD4 and for AKT as loading control. All samples were immunoblotted in the same membrane, but some irrelevant bands were deleted. Note the high molecular weight smears corresponding to polyubiquitinated RTP801. A representative image of three independent experiments is shown. IP = immunoprecipitation; IB = immunoblot.

Article Snippet: Shortly, 1 mM dithiothreitol in 20 mM Tris-HCl pH 7.5, 20 U/ml inorganic pyrophosphatase, 5 mM Mg-ATP, 2,5 μM biotinylated ubiquitin, 0,1 μM His6-tagged recombinant human ubiquitin-activating enzyme E1, His6-tagged recombinant human ubiquitin-conjugating enzyme UbcH5b E2 (Enzo Life Sciences), recombinant human NEDD4 protein (Sigma Aldrich) and N-terminal GST-tagged recombinant human RTP801 full-length protein (Novus Biologicals, Littleton, CO, USA) were added to the reaction buffer, as specified in each condition, and then incubated at 37°C for 90 min. Next, RTP801 was immunoprecipitated using an anti-RTP801 antibody and equal volumes of each sample were analyzed by WB.

Techniques: Ubiquitin Proteomics, Western Blot, Control, Membrane, Cell-Free Assay, Recombinant, Incubation, Immunoprecipitation, Avidin-Biotin Assay, High Molecular Weight, Transfection, Construct, Negative Control

Journal: Oncotarget

Article Title: Loss of NEDD4 contributes to RTP801 elevation and neuron toxicity: implications for Parkinson's disease

doi: 10.18632/oncotarget.11020

Figure Lengend Snippet: a. NEDD4 co-immunoprecipitates with RTP801 in cells exposed to DSP . HEK293 cells were co-transfected with empty vectors pCMS-eGFP and pCI-HA or with pCMS-eGFP-RTP801 and pCI-HA-NEDD4. Twenty-four hours post-transfection cells were exposed to cross-linker DSP for 2 hours at 4°C prior harvesting. RTP801 was immunoprecipitated and the samples were analyzed by Western Blotting. Membranes were probed with anti- NEDD4 and anti-RTP801 antibodies. Representative images are shown of at least three independent experiments. IP = immunoprecipitation; IB = immunoblot. b. . NGF-differentiated PC12 cells were treated with 1 μM epoxomicin for 2 hours. Then, cultures were exposed to DSP at 4 °C for 2 hours prior harvesting. RTP801 immunocomplexes were resolved in a Western Blotting. The membrane was incubated with anti-NEDD4 and anti-RTP801 antibodies. A representative image is shown of at least two independent assays. IP = immunoprecipitation; IB = immunoblot. c. NEDD4 and RTP801 co-localize in neurons. DIV 19 primary rat cortical neurons were transfected with pCI-HA-NEDD4. Forty-eight hours post-transfection, neurons were fixed and stained with anti-RTP801 (in red) and anti-HA (in green). Scale bar, 5 μm.

Article Snippet: Shortly, 1 mM dithiothreitol in 20 mM Tris-HCl pH 7.5, 20 U/ml inorganic pyrophosphatase, 5 mM Mg-ATP, 2,5 μM biotinylated ubiquitin, 0,1 μM His6-tagged recombinant human ubiquitin-activating enzyme E1, His6-tagged recombinant human ubiquitin-conjugating enzyme UbcH5b E2 (Enzo Life Sciences), recombinant human NEDD4 protein (Sigma Aldrich) and N-terminal GST-tagged recombinant human RTP801 full-length protein (Novus Biologicals, Littleton, CO, USA) were added to the reaction buffer, as specified in each condition, and then incubated at 37°C for 90 min. Next, RTP801 was immunoprecipitated using an anti-RTP801 antibody and equal volumes of each sample were analyzed by WB.

Techniques: Transfection, Immunoprecipitation, Western Blot, Membrane, Incubation, Staining

Journal: Oncotarget

Article Title: Loss of NEDD4 contributes to RTP801 elevation and neuron toxicity: implications for Parkinson's disease

doi: 10.18632/oncotarget.11020

Figure Lengend Snippet: a. Ectopic NEDD4 decreases RTP801 protein levels in neuronal PC12 cells. NGF-differentiated PC12 cells were transfected with pCI-HA, pCI-HA-NEDD4 or pCI-HA-NEDD4-C867S constructs. Forty-eight hours post-transfection cultures were harvested and analyzed by Western Blot with anti-NEDD4, anti-RTP801 and anti-α-actin antibodies. Representative immunoblots are shown along with densitometric quantification from at least three independent experiments. ($ endogenous NEDD4, # ectopic NEDD4, * specific band for RTP801). One-way ANOVA with Bonferroni multiple comparison test, * P < 0.05 versus pCI-HA. b. . DIV 8 rat primary cortical neurons were infected with lentiviruses containing the empty vector pHAGE, pHAGE-NEDD4 or pHAGE-NEDD4-C867S inactive mutant. Cell lysates were harvested 4 days later and analyzed by Western Blot with antibodies against RTP801, NEDD4 and α-actin as loading control. The graph represents RTP801 densitometries of at least three independent experiments done in triplicate. One-way ANOVA with Bonferroni multiple comparison test, ** P < 0.01 versus pHAGE. c. Ectopic NEDD4 does not affect RTP801 mRNA levels. DIV 8 cortical neurons were infected with lentiviruses containing the constructs pHAGE, pHAGE-NEDD4 or pHAGE-NEDD4-C867S. RNA was extracted 4 days later, and reverse transcription-qPCR was performed to quantify RTP801 transcripts. Results are displayed as RTP801 mRNA fold change respect to α-actin mRNA levels. The graph shows values (mean ± SEM) of three independent experiments. d. NEDD4 knockdown increases RTP801 protein levels and is detrimental for neurons. DIV 4 cortical neurons were infected with lentiviruses containing a scrambled shRNA (ShCt) or a mix of three shRNA sequences against NEDD4 (ShNEDD4). Six days later, cells were harvested and analyzed by Western Blot. Membranes were incubated with NEDD4, RTP801, P-AKT (S473), AKT and α-spectrin antibodies. The antibody against α-actin was used as loading control. For Western blotting using the α-spectrin antibody the caspase-cleaved fragment (spectrin breakdown product 120, SBDP120) is shown. Representative immunoblots are shown along with RTP801 and P-AKT (S473) densitometries (mean ± SEM) of at least three independent experiments. Student's t -test, * P < 0.05 and ** P < 0.01 versus ShCt. e. NEDD4f/f ;Emx1Cre conditional knockout mice have elevated RTP801 protein levels in the cortex. Cortical lysates of 6-week old mice were analyzed by Western Blotting with anti-NEDD4 and anti-RTP801 antibodies, and then reprobed with anti-α-actin antibody as loading control. Representative immunoblots are shown along with RTP801 densitometries (mean ± SEM) of at least three independent gels. All samples were immunoblotted in the same membrane, but some irrelevant lanes were deleted. (* Specific band for RTP801) Student's t -test, * P < 0.05 versus WT.

Article Snippet: Shortly, 1 mM dithiothreitol in 20 mM Tris-HCl pH 7.5, 20 U/ml inorganic pyrophosphatase, 5 mM Mg-ATP, 2,5 μM biotinylated ubiquitin, 0,1 μM His6-tagged recombinant human ubiquitin-activating enzyme E1, His6-tagged recombinant human ubiquitin-conjugating enzyme UbcH5b E2 (Enzo Life Sciences), recombinant human NEDD4 protein (Sigma Aldrich) and N-terminal GST-tagged recombinant human RTP801 full-length protein (Novus Biologicals, Littleton, CO, USA) were added to the reaction buffer, as specified in each condition, and then incubated at 37°C for 90 min. Next, RTP801 was immunoprecipitated using an anti-RTP801 antibody and equal volumes of each sample were analyzed by WB.

Techniques: Transfection, Construct, Western Blot, Comparison, Infection, Plasmid Preparation, Mutagenesis, Control, Reverse Transcription, Knockdown, shRNA, Incubation, Knock-Out, Membrane

Journal: Oncotarget

Article Title: Loss of NEDD4 contributes to RTP801 elevation and neuron toxicity: implications for Parkinson's disease

doi: 10.18632/oncotarget.11020

Figure Lengend Snippet: a. Ectopic WT NEDD4 protects from RTP801-induced cell death . NGF-differentiated PC12 cells were co-transfected with pCI-HA, pCI-HA-NEDD4 or pCI-HA-NEDD4-C867S constructs, together with either pCMS-eGFP or pCMS-eGFP-RTP801. Cells were fixed 24 hours later and cell survival (eGFP+ cells) scored under fluorescence microscopy. The graph represents mean ± SEM of at least three independent experiments in quadruplicates. One-way ANOVA with Bonferroni multiple comparison test, *** P < 0.001 versus pCI-HA/pCMS-eGFP, ## P < 0.01 versus pCI-HA/pCMS-eGFP-RTP801. b. . NGF-differentiated PC12 cells were co-transfected with pCI-HA or pCI-HA-NEDD4 together with pCMS-eGFP, pCMS-eGFP-RTP801 or pCMS-eGFP-RTP801-KR. Twenty-four hours later, cells were fixed and eGFP+ surviving cells scored using fluorescence microscopy. The graph represents mean ± SEM of at least three independent experiments in quadruplicates. One-way ANOVA with Bonferroni multiple comparison test, *** P < 0.001 versus pCI-HA/pCMS-eGFP and # P < 0.05 versus pCI-HA/pCMS-eGFP-RTP801.

Article Snippet: Shortly, 1 mM dithiothreitol in 20 mM Tris-HCl pH 7.5, 20 U/ml inorganic pyrophosphatase, 5 mM Mg-ATP, 2,5 μM biotinylated ubiquitin, 0,1 μM His6-tagged recombinant human ubiquitin-activating enzyme E1, His6-tagged recombinant human ubiquitin-conjugating enzyme UbcH5b E2 (Enzo Life Sciences), recombinant human NEDD4 protein (Sigma Aldrich) and N-terminal GST-tagged recombinant human RTP801 full-length protein (Novus Biologicals, Littleton, CO, USA) were added to the reaction buffer, as specified in each condition, and then incubated at 37°C for 90 min. Next, RTP801 was immunoprecipitated using an anti-RTP801 antibody and equal volumes of each sample were analyzed by WB.

Techniques: Transfection, Construct, Fluorescence, Microscopy, Comparison

Journal: Oncotarget

Article Title: Loss of NEDD4 contributes to RTP801 elevation and neuron toxicity: implications for Parkinson's disease

doi: 10.18632/oncotarget.11020

Figure Lengend Snippet: a. NEDD4 protein levels are diminished in 6-OHDA-treated neuronal PC12 cells. NGF-differentiated PC12 cells were exposed to 100 μM 6-OHDA for 16 hours prior harvesting. Cell lysates were analyzed by Western Blot with antibodies against NEDD4, RTP801, as well as α-actin antibody as loading control. Representative immunoblots are shown along with densitometries represented as mean ± SEM of at least three independent experiments. Student's t -test, ** P < 0.01 and *** P < 0.001 versus Ut (Untreated). b. NEDD4 mRNA levels are not modified in 6-OHDA-treated neuronal PC12 cells. NGF-differentiated PC12 cells were exposed to 100 μM 6-OHDA for 8 hours. RNA was extracted and reverse transcription-qPCR was performed. The graphs show values (mean ± SEM) of three independent experiments. Student's t -test, *** P < 0.001 versus ut (untreated). c. NEDD4 is cleaved by caspases after 6-OHDA exposure. Neuronal PC12 cells were treated with 100 μM Z-VAD-FMK pan-caspase inhibitor for 1 hour prior to 6-OHDA exposure. Sixteen hours later, cultures were harvested and analyzed by Western Blot. Membranes were incubated with antibodies against NEDD4 and α-spectrin as well as an α-actin antibody as loading control. Graphs represent mean ± SEM of NEDD4 and caspase-cleaved α-spectrin fragment (spectrin breakdown product 120 KDa, SBDP 120) densitometric quantification of at least three independent experiments. One-way ANOVA with Bonferroni multiple comparison test, *** P < 0.001 versus Untreated/Ct and # P < 0.05, ### P < 0.001 versus 100 μM 6-OHDA/Ct. d. NEDD4 is cleaved by calpains after 6-OHDA exposure. Neuronal PC12 cells were treated with 1 μM ALLN calpain inhibitor for 1 hour prior to 6-OHDA exposure. Sixteen hours later, cultures were harvested and subjected to Western Blot. Membranes were incubated with anti-NEDD4 and anti-α-spectrin antibodies and with anti-α-actin as loading control. Graphs represent mean ± SEM of NEDD4 and calpain-cleaved α-spectrin fragment (spectrin breakdown product 145 KDa, SBDP 145) densitometric quantification of at least three independent experiments. One-way ANOVA with Newman-Keuls multiple comparison test, ** P < 0.01, *** P < 0.001 versus Untreated/Ct and # P < 0.05, ### P < 0.001 versus 100 μM 6-OHDA/Ct.

Article Snippet: Shortly, 1 mM dithiothreitol in 20 mM Tris-HCl pH 7.5, 20 U/ml inorganic pyrophosphatase, 5 mM Mg-ATP, 2,5 μM biotinylated ubiquitin, 0,1 μM His6-tagged recombinant human ubiquitin-activating enzyme E1, His6-tagged recombinant human ubiquitin-conjugating enzyme UbcH5b E2 (Enzo Life Sciences), recombinant human NEDD4 protein (Sigma Aldrich) and N-terminal GST-tagged recombinant human RTP801 full-length protein (Novus Biologicals, Littleton, CO, USA) were added to the reaction buffer, as specified in each condition, and then incubated at 37°C for 90 min. Next, RTP801 was immunoprecipitated using an anti-RTP801 antibody and equal volumes of each sample were analyzed by WB.

Techniques: Western Blot, Control, Modification, Reverse Transcription, Incubation, Comparison

Journal: Oncotarget

Article Title: Loss of NEDD4 contributes to RTP801 elevation and neuron toxicity: implications for Parkinson's disease

doi: 10.18632/oncotarget.11020

Figure Lengend Snippet: a. Ectopic NEDD4 WT partially prevents from 6-OHDA-induced cell death. NGF-differentiated PC12 cells were co-transfected with pCI-HA/pCMS-eGFP, pCI-HA-NEDD4/pCMS-eGFP or pCI-HA-NEDD4-C867S/pCMS-eGFP vectors with a 4:1 ratio. Thirty-two hours later, cell cultures were exposed to 100 μM 6-OHDA for 16 hours. Then, eGFP+ surviving cells were scored under fluorescence microscopy. The graph shows mean ± SEM of at least three independent experiments done in quadruplicate. One-way ANOVA with Bonferroni multiple comparison test, *** P < 0.001 versus Untreated/pCI-HA and ## P < 0.01 versus 100 μM 6-OHDA /pCI-HA. b. Ectopic NEDD4 reduces RTP801 elevation after 6-OHDA exposure. NGF-differentiated PC12 cells were transfected with pCI-HA, pCI-HA-NEDD4 or pCI-HA-NEDD4-C867S. Thrity-two hours later, cell cultures were exposed to 100 μM 6-OHDA for 16 hours and cell lysates were analyzed by Western Blot. Membranes were incubated with antibodies against NEDD4, RTP801 and α-actin as loading control (* endogenous NEDD4, # ectopic NEDD4). Low signal of ectopic NEDD4 maybe due low sensitivity of NEDD4 antibody towards the human NEDD4 protein, as compared to endogenous rat NEDD4. Representative immunoblots are shown along with RTP801 densitometric normalized quantification (mean ± SEM) from at least three independent experiments. One-way ANOVA with Bonferroni multiple comparison test, * P < 0.05 versus Untreated/pCI-HA, # P < 0.05 versus 100 μM 6-OHDA/pCI-HA.

Article Snippet: Shortly, 1 mM dithiothreitol in 20 mM Tris-HCl pH 7.5, 20 U/ml inorganic pyrophosphatase, 5 mM Mg-ATP, 2,5 μM biotinylated ubiquitin, 0,1 μM His6-tagged recombinant human ubiquitin-activating enzyme E1, His6-tagged recombinant human ubiquitin-conjugating enzyme UbcH5b E2 (Enzo Life Sciences), recombinant human NEDD4 protein (Sigma Aldrich) and N-terminal GST-tagged recombinant human RTP801 full-length protein (Novus Biologicals, Littleton, CO, USA) were added to the reaction buffer, as specified in each condition, and then incubated at 37°C for 90 min. Next, RTP801 was immunoprecipitated using an anti-RTP801 antibody and equal volumes of each sample were analyzed by WB.

Techniques: Transfection, Fluorescence, Microscopy, Comparison, Western Blot, Incubation, Control

Journal: Oncotarget

Article Title: Loss of NEDD4 contributes to RTP801 elevation and neuron toxicity: implications for Parkinson's disease

doi: 10.18632/oncotarget.11020

Figure Lengend Snippet: DIV 5 primary rat cortical neurons were infected with lentiviruses containing a scrambled shRNA (ShCt) or a shRNA against RTP801 (ShRTP801). Two days later, neurons were transduced with a mix of three shRNA sequences against NEDD4 (ShNEDD4) or the corresponding control shRNA (ShCt). Cell lysates were analyzed 4 days later by Western Blot. Membranes were incubated with RTP801, NEDD4, P-AKT (S473), AKT, P-S6 (S235/236), ERK1/2 and α-spectrin antibodies, and with α-actin antibody as loading control. For α-spectrin the caspase-cleaved fragment (spectrin breakdown product 120, SBDP120) is shown. Representative immunoblots are shown along with densitometries (mean ± SEM) of at least two independent experiments done in triplicate. One-way ANOVA with Newman-Keuls multiple comparison test, * P < 0.05, ** P < 0.01 versus ShCt/ShCt and # P < 0.05, ## P < 0.01 versus ShCt/ShNEDD4.

Article Snippet: Shortly, 1 mM dithiothreitol in 20 mM Tris-HCl pH 7.5, 20 U/ml inorganic pyrophosphatase, 5 mM Mg-ATP, 2,5 μM biotinylated ubiquitin, 0,1 μM His6-tagged recombinant human ubiquitin-activating enzyme E1, His6-tagged recombinant human ubiquitin-conjugating enzyme UbcH5b E2 (Enzo Life Sciences), recombinant human NEDD4 protein (Sigma Aldrich) and N-terminal GST-tagged recombinant human RTP801 full-length protein (Novus Biologicals, Littleton, CO, USA) were added to the reaction buffer, as specified in each condition, and then incubated at 37°C for 90 min. Next, RTP801 was immunoprecipitated using an anti-RTP801 antibody and equal volumes of each sample were analyzed by WB.

Techniques: Infection, shRNA, Transduction, Control, Western Blot, Incubation, Comparison

Journal: Cell Death & Disease

Article Title: Parkin loss of function contributes to RTP801 elevation and neurodegeneration in Parkinson's disease

doi: 10.1038/cddis.2014.333

Figure Lengend Snippet: RTP801 is poly-ubiquitinated by parkin E3 ligase and degraded by the proteasome. ( a ) RTP801 is degraded by the proteasome. HEK293 cells were exposed for 4 h to 1 μ M epoxomycin or 50 μ M chloroquine, and cell lysates were analyzed by western immunoblotting for RTP801 and α -actin (loading control). ut = untreated. ( b ) RTP801 is poly-ubiquitinated by parkin prior to proteasomal degradation. HEK293 cells were co-transfected with pCMS-eGFP RTP801, HA-ubiquitin and one of the pRK5-myc constructs, either empty or containing parkin WT or parkin ΔR2. Twenty-four hours later, cultures were exposed to epoxomycin for 3 h prior to harvesting. RTP801 was immunoprecipitated (IP) and immunocomplexes along with whole-cell lysates were analyzed by western immunoblotting (IB). Membranes were probed for HA, myc and α -actin as a loading control. Membranes were reprobed with anti-RTP801 antibody to confirm that RTP801 was immunoprecipitated. Non-specific mouse immunoglobulins were used as negative controls (m IgG). (HMW Ub-RTP801, high molecular weight ubiquitinated RTP801) ( c ) Parkin ubiquitinates RTP801 in a cell-free in vitro system. Recombinant parkin E3 ligase (active full-length), truncated parkin (inactive), GST-RTP801, and UbcH7 E2 enzyme, were mixed and incubated as indicated along with biotinylated ubiquitin, E1 enzyme, and ATP. RTP801 was immunoprecipitated, and immunocomplexes resolved in a western blot. The membrane was incubated with Avidin/Biotin and then incubated with chemiluminiscent peroxidase substrate solution (upper panel). The same membrane was reprobed with an anti-RTP801 antibody (lower panel). (HMW Ub-RTP801, high molecular weight ubiquitinated RTP801) ( d ) Ectopic parkin physically interacts with ectopic RTP801. HEK293 cells were co-transfected either with the pCMS-eGFP and pEGFP-C2 empty vectors or pCMS-eGFP RTP801 along with pEGFP-C2-parkin. Twenty-four hours later, cultures were treated with the cross-linking agent DSP for 2 h at 4°C, prior to harvesting. After RTP801 immunoprecipitation, the samples were analyzed by western immunoblotting for parkin to detect the interaction, and for RTP801 as an IP control. ( e and f ) Reciprocal co-immunoprecipitation indicates interaction of endogenous parkin and RTP801 in cells. Neuronal PC12 cells were treated with or without MG132 for 5 h as indicated, lysed and subjected to immunoprecipitation (IP) with control antibody (GFP), and either ( e ) anti-parkin or ( f ) anti-RTP801 antibodies. Immunoprecipitates were analyzed by western immunoblotting with anti-parkin and anti-RTP801 as indicated. In ( e ) all samples were analyzed on the same blot, but irrelevant intervening lanes were removed. In the right panel ( f ), the membrane was probed with a light chain-specific secondary antibody to diminish overlap of signal with co-immunoprecipitated RTP801. In each panel ( a , b , c , d , e and f ), a representative western blot is shown from a pool of at least two independent experiments. Specific bands are pointed out by arrows

Article Snippet: Briefly, (20 mM Tris-HCl; pH 7.5, 1 mM dithiothreitol, 20 U/ml inorganic pyrophosphatase, 5 mM Mg-ATP, 0.1 μ M His 6 -tagged recombinant human ubiquitin-activating enzyme E1, 2.5 μ M biotinylated ubiquitin), His 6 -tagged recombinant human ubiquitin-conjugating enzyme UbcH7 E2 (Enzo Life Biosciences), His 6 -tagged recombinant human parkin full-lenght (Merck Millipore), N-terminal GST-tagged recombinant human RTP801 full-length (Novus Biologicals, Cambridge, UK), and GST-tagged recombinant human truncated and inactive parkin (1-387) (Novus Biologicals), were added to the reaction buffer, as referred in each condition, and then incubated at 37 °C for 90 min. RTP801 was immunoprecipitated as described above, and equal volumes of each sample were subjected to WB.

Techniques: Western Blot, Transfection, Construct, Immunoprecipitation, Molecular Weight, In Vitro, Recombinant, Incubation, Membrane, Avidin-Biotin Assay