Journal: Cell reports

Article Title: HAPSTR1 localizes HUWE1 to the nucleus to limit stress signaling pathways

doi: 10.1016/j.celrep.2023.112496

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-REDD1 (DDIT4) , Proteintech , Cat#10638–1-AP; RRID:AB_2245711.

Techniques: Recombinant, Protease Inhibitor, Western Blot, SYBR Green Assay, Conjugation Assay, Ubiquitin Proteomics, Bicinchoninic Acid Protein Assay, Luciferase, Viability Assay, Control, Knock-In, Expressing, Sequencing, Software

Journal: Molecular Biology of the Cell

Article Title: Nuclear factor of activated T-cell c3 inhibition of mammalian target of rapamycin signaling through induction of regulated in development and DNA damage response 1 in human intestinal cells

doi: 10.1091/mbc.E12-01-0037

Figure Lengend Snippet: PMA/Io-mediated induction of REDD1 expression was attenuated by CsA, a potent calcineurin inhibitor in intestinal cells. (A) HT29 cells were treated with PMA (100 nM) plus Io (2.5 μM) over a time course. Total protein was extracted from cells, resolved by SDS–PAGE, transferred to a PVDF membrane, and probed with anti-REDD1 and anti–β-actin antibodies. (B) HT29 cells were pretreated with actinomycin D (10 μg/ml) for 30 min; this was followed by treatment with a combination of PMA (100 nM) plus Io (2.5 μM) with actinomycin D (10 μg/ml) for 1.5 h. (C) HT29 cells were pretreated with CsA for 30 min; this was followed by treatment with a combination of PMA (100 nM) plus Io (2.5 μM) with CsA for 1.5 h. Total RNA was extracted, and REDD1 mRNA levels were determined by real-time RT-PCR. (Data represent mean ± SD; *, p < 0.01 vs. control; #, p < 0.01 vs. PMA/Io alone as determined by ANOVA.) (D) HT29 cells were pretreated with CsA for 30 min; this was followed by treatment with a combination of PMA (100 nM) plus Io (2.5 μM) with CsA for 1.5 h. Total protein was extracted from cells, resolved by SDS–PAGE, and subjected to Western blotting using anti-REDD1, anti–c-Myc, anti–phospho-S6 (pS235/236), anti-S6, anti–phospho-4E-BP-1 (pT37/46), anti–4E-BP-1 and anti–β-actin antibodies. (E) Caco-2, SW480, and HCT116 cells were treated with PMA (100 nM) plus Io (2.5 μM) over a time course. Total protein was extracted from cells, resolved by SDS–PAGE, transferred to a PVDF membrane, and probed with anti-REDD1, anti–β-actin, anti–phospho-S6, and anti-S6 antibodies.

Article Snippet: Rabbit polyclonal anti-REDD1 antibody was from Proteintech Group (Chicago, IL).

Techniques: Expressing, SDS Page, Membrane, Quantitative RT-PCR, Control, Western Blot

Journal: Molecular Biology of the Cell

Article Title: Nuclear factor of activated T-cell c3 inhibition of mammalian target of rapamycin signaling through induction of regulated in development and DNA damage response 1 in human intestinal cells

doi: 10.1091/mbc.E12-01-0037

Figure Lengend Snippet: Knockdown of NFATc3-attenuated PMA/Io induced REDD1 expression in HT29 cells. (A) HT29 cells were transfected with control siRNA or siRNA specifically targeting NFATc1, c2, c3, or c4. After a 46-h incubation, transfected cells were treated with PMA (100 nM) plus Io (2.5 μM) for an additional 1.5 h. Cells were lysed, and Western blot analysis was performed using antibodies against REDD1 and β-actin. (B) HT29 cells were transfected with control siRNA or siRNA targeting NFATc1, c2, c3, or c4. After a 46-h incubation, total RNA was extracted, and real-time RT-PCR was performed for analysis of NFATc1, NFATc2, NFATc3, and NFATc4 mRNA expression. (Data represent mean ± SD; * p < 0.01 vs control as determined by two-sample t tests.) (C) HT29 cells were transfected with control siRNA or siRNA targeting NFATc1, c2, c3, or c4. After a 46-h incubation, total protein was extracted, and Western blotting was performed for analysis of NFATc1, NFATc2, NFATc3, and NFATc4 protein expression.

Article Snippet: Rabbit polyclonal anti-REDD1 antibody was from Proteintech Group (Chicago, IL).

Techniques: Knockdown, Expressing, Transfection, Control, Incubation, Western Blot, Quantitative RT-PCR

Journal: Molecular Biology of the Cell

Article Title: Nuclear factor of activated T-cell c3 inhibition of mammalian target of rapamycin signaling through induction of regulated in development and DNA damage response 1 in human intestinal cells

doi: 10.1091/mbc.E12-01-0037

Figure Lengend Snippet: NFATc3 regulated REDD1 expression in HT29, Caco-2, HCT116, and SW480 cells. (A) HT29 cells were transfected with control vector or NFATc3 (left) or control siRNA or siRNA targeting NFATc3 (right). After a 48-h incubation, REDD1, NFATc3, β-actin, phospho-mTOR (pS2448), mTOR, phospho-S6 (pS235/236), and S6 expression was determined by Western blotting. (B and C) HT29 cells were transfected with control vector or NFATc3 plasmid (B) or transfected with control siRNA or siRNA targeting NFATc3 (C). After a 48-h incubation, total RNA was extracted and REDD1 and NFATc3 mRNA levels were determined by real-time RT-PCR. (Data represent mean ± SD; * p < 0.01 vs. control as determined by two-sample t tests.) (D and E) Caco-2, HCT116, and SW480 cells were transfected with control vector or NFATc3 (D) or transfected with control siRNA or siRNA targeting NFATc3 (E). After a 48-h incubation, REDD1, NFATc3, β-actin, phospho-S6 (pS235/236), and S6 expression was determined by Western blotting.

Article Snippet: Rabbit polyclonal anti-REDD1 antibody was from Proteintech Group (Chicago, IL).

Techniques: Expressing, Transfection, Control, Plasmid Preparation, Incubation, Western Blot, Quantitative RT-PCR

Journal: Molecular Biology of the Cell

Article Title: Nuclear factor of activated T-cell c3 inhibition of mammalian target of rapamycin signaling through induction of regulated in development and DNA damage response 1 in human intestinal cells

doi: 10.1091/mbc.E12-01-0037

Figure Lengend Snippet: NFATc3 is a transcriptional activator of REDD1 in HT29 cells. (A and B) HT29 cells were transfected with a construct containing REDD1 promoter sequence (−2931/−97) alone (A) or together with either control plasmid or an NFATc3 plasmid (B). At 43-h posttransfection, cells were treated with or without PMA/Io for 5 h. Cells were harvested, and luciferase activity was assayed. All results were normalized for transfection efficiency using the pRL-Tk-luc plasmid (Promega). (Data shown are mean ± SD; * p < 0.05, PMA/Io vs. control, or NFATc3 alone or PMA/Io plus vector vs. vector alone; #, p < 0.05, PMA/Io plus CsA vs. control or PMA/Io plus NFATc3 vs. NFATc3 alone, as determined by ANOVA.) (C) HT29 cells were subjected to ChIP assay; soluble chromatin was prepared from HT29 cells treated with PMA/Io for 1 h and immunoprecipitated with NFATc3 antibody or IgG. Total (Input) and immunoprecipitated DNAs or H 2 O (negative control) were then PCR-amplified using primer pairs covering −890/−97 within the human REDD1 promoter.

Article Snippet: Rabbit polyclonal anti-REDD1 antibody was from Proteintech Group (Chicago, IL).

Techniques: Transfection, Construct, Sequencing, Control, Plasmid Preparation, Luciferase, Activity Assay, Immunoprecipitation, Negative Control, Amplification

Journal: Molecular Biology of the Cell

Article Title: Nuclear factor of activated T-cell c3 inhibition of mammalian target of rapamycin signaling through induction of regulated in development and DNA damage response 1 in human intestinal cells

doi: 10.1091/mbc.E12-01-0037

Figure Lengend Snippet: NFATc3/REDD1 regulation of c-Myc expression. (A) Total protein from HT29 cells stably transfected with control or REDD1 shRNA was extracted and analyzed by Western blotting using anti-REDD1, anti-S6, anti–phospho-S6 (pS235/236), and anti–β-actin antibodies. (B) HT29 cells were transfected with control siRNA or siRNA targeting NFATc3. After a 46.5-h incubation, transfected cells were treated with PMA (100 nM) plus Io (2.5 μM) for an additional 1.5 h. Cells were lysed, and Western blot analysis was performed using antibodies against c-Myc, REDD1, NFATc3, and β-actin. (C) Caco-2 and HCT116 cells were transfected with control vector or NFATc3 plasmid. After a 48-h incubation, c-Myc and NFATc3 expression was determined by Western blotting. β-actin was blotted to confirm equal loading.

Article Snippet: Rabbit polyclonal anti-REDD1 antibody was from Proteintech Group (Chicago, IL).

Techniques: Expressing, Stable Transfection, Transfection, Control, shRNA, Western Blot, Incubation, Plasmid Preparation

Journal: Molecular Biology of the Cell

Article Title: Nuclear factor of activated T-cell c3 inhibition of mammalian target of rapamycin signaling through induction of regulated in development and DNA damage response 1 in human intestinal cells

doi: 10.1091/mbc.E12-01-0037

Figure Lengend Snippet: NFATc3 and REDD1 regulation of MUC2 mRNA expression. (A) HT29 cells were pretreated with CsA for 30 min; this was followed by treatment with the combination of PMA/Io for 1.5 h. (B) HT29 cells were transfected with control siRNA or siRNA targeting NFATc3 (left panel) or transfected with control vector or NFATc3 plasmid (right panel). Transfected cells were incubated for 48 h. (C) HT29 cells were transfected with control siRNA or siRNA targeting NFATc3. After a 46-h incubation, transfected cells were treated with PMA/Io for an additional 1.5 h. (D) HT29 cells, stably transfected with control or REDD1 shRNA, were treated with PMA/Io for 1.5 h. (E) HT29 cells were transfected with control siRNA or siRNA targeting TSC2 and incubated for 48 h. Total RNA was extracted, and MUC2 mRNA levels were determined by real-time RT-PCR (A–E). (Data represent mean ± SD; * p < 0.01 vs. control or control siRNA or control shRNA or vector alone; # p < 0.01 vs. PMA/Io alone as determined by ANOVA.) Total protein was extracted and subjected to Western blotting using anti-TSC2, anti–phospho-S6 (pS235/236), anti-S6, and anti–β-actin antibodies (E).

Article Snippet: Rabbit polyclonal anti-REDD1 antibody was from Proteintech Group (Chicago, IL).

Techniques: Expressing, Transfection, Control, Plasmid Preparation, Incubation, Stable Transfection, shRNA, Quantitative RT-PCR, Western Blot

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: Goat anti-mouse and anti-rabbit secondary antibodies conjugated to Alexa 488 or Alexa 568 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). pCMS-eGFP-RTP801 and pCMS-eGFP-RTP801 KR constructs were generated as previously described [ , ]. pCI-HA-NEDD4 and pcDNA3 HA-ubiquitin were purchased from Addgene (Cambridge, MA, USA).

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: Goat anti-mouse and anti-rabbit secondary antibodies conjugated to Alexa 488 or Alexa 568 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). pCMS-eGFP-RTP801 and pCMS-eGFP-RTP801 KR constructs were generated as previously described [ , ]. pCI-HA-NEDD4 and pcDNA3 HA-ubiquitin were purchased from Addgene (Cambridge, MA, USA).

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: Goat anti-mouse and anti-rabbit secondary antibodies conjugated to Alexa 488 or Alexa 568 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). pCMS-eGFP-RTP801 and pCMS-eGFP-RTP801 KR constructs were generated as previously described [ , ]. pCI-HA-NEDD4 and pcDNA3 HA-ubiquitin were purchased from Addgene (Cambridge, MA, USA).

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: Goat anti-mouse and anti-rabbit secondary antibodies conjugated to Alexa 488 or Alexa 568 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). pCMS-eGFP-RTP801 and pCMS-eGFP-RTP801 KR constructs were generated as previously described [ , ]. pCI-HA-NEDD4 and pcDNA3 HA-ubiquitin were purchased from Addgene (Cambridge, MA, USA).

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: Goat anti-mouse and anti-rabbit secondary antibodies conjugated to Alexa 488 or Alexa 568 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). pCMS-eGFP-RTP801 and pCMS-eGFP-RTP801 KR constructs were generated as previously described [ , ]. pCI-HA-NEDD4 and pcDNA3 HA-ubiquitin were purchased from Addgene (Cambridge, MA, USA).

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: Goat anti-mouse and anti-rabbit secondary antibodies conjugated to Alexa 488 or Alexa 568 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). pCMS-eGFP-RTP801 and pCMS-eGFP-RTP801 KR constructs were generated as previously described [ , ]. pCI-HA-NEDD4 and pcDNA3 HA-ubiquitin were purchased from Addgene (Cambridge, MA, USA).

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: Goat anti-mouse and anti-rabbit secondary antibodies conjugated to Alexa 488 or Alexa 568 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). pCMS-eGFP-RTP801 and pCMS-eGFP-RTP801 KR constructs were generated as previously described [ , ]. pCI-HA-NEDD4 and pcDNA3 HA-ubiquitin were purchased from Addgene (Cambridge, MA, USA).

Techniques: Infection, shRNA, Transduction, Control, Western Blot, 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. 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: Goat anti-mouse and anti-rabbit secondary antibodies conjugated to Alexa 488 or Alexa 568 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). pCMS-eGFP-RTP801 and pCMS-eGFP-RTP801 KR constructs were generated as previously described [ , ]. pCI-HA-NEDD4 and pcDNA3 HA-ubiquitin were purchased from Addgene (Cambridge, MA, USA).

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: Goat anti-mouse and anti-rabbit secondary antibodies conjugated to Alexa 488 or Alexa 568 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). pCMS-eGFP-RTP801 and pCMS-eGFP-RTP801 KR constructs were generated as previously described [ , ]. pCI-HA-NEDD4 and pcDNA3 HA-ubiquitin were purchased from Addgene (Cambridge, MA, USA).

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: Goat anti-mouse and anti-rabbit secondary antibodies conjugated to Alexa 488 or Alexa 568 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). pCMS-eGFP-RTP801 and pCMS-eGFP-RTP801 KR constructs were generated as previously described [ , ]. pCI-HA-NEDD4 and pcDNA3 HA-ubiquitin were purchased from Addgene (Cambridge, MA, USA).

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: Goat anti-mouse and anti-rabbit secondary antibodies conjugated to Alexa 488 or Alexa 568 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). pCMS-eGFP-RTP801 and pCMS-eGFP-RTP801 KR constructs were generated as previously described [ , ]. pCI-HA-NEDD4 and pcDNA3 HA-ubiquitin were purchased from Addgene (Cambridge, MA, USA).

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: Goat anti-mouse and anti-rabbit secondary antibodies conjugated to Alexa 488 or Alexa 568 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). pCMS-eGFP-RTP801 and pCMS-eGFP-RTP801 KR constructs were generated as previously described [ , ]. pCI-HA-NEDD4 and pcDNA3 HA-ubiquitin were purchased from Addgene (Cambridge, MA, USA).

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: Goat anti-mouse and anti-rabbit secondary antibodies conjugated to Alexa 488 or Alexa 568 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). pCMS-eGFP-RTP801 and pCMS-eGFP-RTP801 KR constructs were generated as previously described [ , ]. pCI-HA-NEDD4 and pcDNA3 HA-ubiquitin were purchased from Addgene (Cambridge, MA, USA).

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: Goat anti-mouse and anti-rabbit secondary antibodies conjugated to Alexa 488 or Alexa 568 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). pCMS-eGFP-RTP801 and pCMS-eGFP-RTP801 KR constructs were generated as previously described [ , ]. pCI-HA-NEDD4 and pcDNA3 HA-ubiquitin were purchased from Addgene (Cambridge, MA, USA).

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