Journal: Apoptosis : an international journal on programmed cell death

Article Title: Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes

doi: 10.1007/s10495-013-0922-7

Figure Lengend Snippet: (a) – Phase contrast light microscopy of proliferating myoblasts and myotubes formed on day 6 of differentiation. (b) - WB analysis of specific protein marker expression during C2C12 cell differentiation assessed with antibodies against myogenin, SERCA1, Cav3, and Cav1. Actin expression levels are shown as controls for protein load. (c) – Myotubes and reserve cells were separated at days 4 (4d) and 6 (6d) of differentiation, as shown in the schematic representation and described under “Experimental Procedures”, and analyzed by WB with antibodies specific for myogenin, SERCA1, Cav3, and Cav1. The abbreviations used for the cell types are M - proliferating myoblasts; T – myotubes; R - reserve cells.

Article Snippet: Cell Culture and incubations C2C12 mouse skeletal muscle cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Light Microscopy, Marker, Expressing, Cell Differentiation

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes

doi: 10.1007/s10495-013-0922-7

Figure Lengend Snippet: (a) - Proliferating myoblasts (day 0) and differentiating C2C12 cells were harvested on days 2 - 6 after the onset of differentiation and analyzed by SDS-PAGE and WB analysis with antibodies against Hsp70, Bcl-2, Bax, Bak, and Bad. (b) – Analysis of these proteins separately in myoblasts (M), myotubes (T) and reserve cells (R) along with cell type-specific protein markers Pax7 and MyoD at days 4 (4d) and day 6 (6d) of differentiation. (c) Densitometry analysis of data from (b).

Article Snippet: Cell Culture and incubations C2C12 mouse skeletal muscle cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD).

Techniques: SDS Page

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes

doi: 10.1007/s10495-013-0922-7

Figure Lengend Snippet: (a) – Sequence specificity of SERCA2A and SERCA2B in the C-terminal part. (b) – WB analysis of protein expression during C2C12 cell differentiation assessed with antibodies against SERCA2. (c) - Densitometry analysis of WB data of SERCA2 level in GM and in DM after 6 days of differentiation from three independent experiments.(d) – Analysis of SERCA2 isoforms separately in myoblasts (M), myotubes (T) and reserve cells (R): myotubes and reserve cells were separated as described under “Experimental Procedures” at day 6 of differentiation and analyzed by WB with antibodies specific for SERCA2 and SERCA2B (polyclonal custom antibody raised against the C-terminal peptide underlined in panel A). (e) - Capillary LC-LTQ-FT-MS/MS spectrum of the C-terminal tryptic peptide of SERCA2A, identified in the tryptic digests of C2C12 cell lysates.

Article Snippet: Cell Culture and incubations C2C12 mouse skeletal muscle cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Sequencing, Expressing, Cell Differentiation, Tandem Mass Spectroscopy

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes

doi: 10.1007/s10495-013-0922-7

Figure Lengend Snippet: Phase contrast light microscopy of differentiated C2C12 cells prior to (a) and after exposure to 0.5 (b), 1 (c), 2 (d), and 4 mM H2O2 (e) for 4 h or 2 h (f) as described under “Experimental Procedures”.

Article Snippet: Cell Culture and incubations C2C12 mouse skeletal muscle cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Light Microscopy

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes

doi: 10.1007/s10495-013-0922-7

Figure Lengend Snippet: Differentiated C2C12 cells were incubated without (control) or with 4 mM H2O2 for different times as indicated (a) or incubated overnight with smaller concentrations of H2O2 (b), and adherent cells were analyzed by WB with anti-SERCA1, anti-Cav3 and anti-Bcl-2 antibodies. Actin expression levels are presented for control of protein load. (c) Differentiated C2C12 cells were incubated in the absence or in the presence of 4 mM H2O2 for 4 h, and expression levels of SERCA1 and Bcl-2 were analyzed by WB separately for adherent and detached cells.

Article Snippet: Cell Culture and incubations C2C12 mouse skeletal muscle cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Incubation, Control, Expressing

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes

doi: 10.1007/s10495-013-0922-7

Figure Lengend Snippet: (a) – Detached cells obtained after different treatment were harvested as depicted in the inserted scheme and analyzed by WB for Cav3, Bcl-2, and actin. (b) – Differentiated C2C12 cells treated as above were lysed, digested with trypsin, and submitted to a “shotgun” capLC-LTQ-FT-MS/MS analysis coupled to a protein database search, followed by a quantification of peptide TIC values for selected proteins as described under “Experimental Procedures”. Protein IDs in the IPI (mouse) database: SERCA1, 00311654; SERCA2, 00468900; Myosin light chain, skeletal muscle, 00224549; Myosin 3, 00380895; Myosin 9, 00123181; Troponin c, slow-twitch skeletal muscle, 00113712; Tubulin beta, 00117352. (Note: error bars on logarithmic scale).

Article Snippet: Cell Culture and incubations C2C12 mouse skeletal muscle cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Tandem Mass Spectroscopy

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes

doi: 10.1007/s10495-013-0922-7

Figure Lengend Snippet: Differentiated C2C12 cells were incubated without (control) or in the presence of 4 mM of H2O2 for 2 h as shown in the schematic representation, and myotubes and reserve cells were separated by mild trypsinization as described under “Experimental Procedure”. (a) – Scheme of experiment. (b) – Expression profiles of Bcl-2, SERCA1, Cav3, and Pax7 in myotubes and reserve cells analyzed by WB after incubation without (control) or with 4 mM H2O2 for 2 h. (c) - Bcl-2 levels in non-treated myotubes (control) and myotubes remaining attached to the dish after incubation with 4 mM H2O2 for 2 h, collected by mild trypsinization, and analyzed by a densitometry analysis of the respective blots presented in panel B.

Article Snippet: Cell Culture and incubations C2C12 mouse skeletal muscle cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Incubation, Control, Expressing

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes

doi: 10.1007/s10495-013-0922-7

Figure Lengend Snippet: Proliferating C2C12 myoblasts were transfected with either empty vector or Bcl-2 expression vector for 24 h before the onset of differentiation. (a) - Cells detached from the dishes at days 2, 4, and 6 after the initiation of differentiation were harvested and analyzed by WB for Bcl-2, SERCA1, Cav3 and caspase 9. (b) - Myotubes and reserve cells remaining attached to the dishes at day 6 after initiation of differentiation were separated via differential trypsinization as described under “Experimental Procedures”, and analyzed by WB with anti-Bcl-2 and anti-Bax antibodies. (c) – Control and Bcl-2 transfected C2C12 cells at day 6 after initiation of differentiation were incubated without or with 4 mM H2O2 for 2 hours, followed by WB analysis for Cav3, Bcl-2, and caspase 9 of both adherent and detached cells. (d) - Densitometry quantification of Cav-3 on the blots (marker of myotubes) shown in panel C, normalized to the level of Cav-3 in Bcl-2 transfected cells, remaining adherent to the dish after exposure to 4 mM H2O2 for 2 hours.

Article Snippet: Cell Culture and incubations C2C12 mouse skeletal muscle cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Transfection, Plasmid Preparation, Expressing, Control, Incubation, Marker

Journal: BMC Biochemistry

Article Title: Hepatoma-derived growth factor and nucleolin exist in the same ribonucleoprotein complex

doi: 10.1186/1471-2091-14-2

Figure Lengend Snippet: Mass spectrometry analysis of purified HDGF protein complexes. HDGF interaction partners were isolated using Strep-Tactin®-MacroPrep matrices as affinity resin to purify HDGFStrep-tag proteins. Trapped proteins were digested by trypsin and peptides analyzed by ESI-MS/MS. ( A ) Flow chart of the experiment. ( B ) Venn diagramm showing the overlap between HDGFStrep-tag and GFPStrep-tag interactome. ( C ) Pie chart of molecular functions of HDGFStrep-tag interaction partners (see also Additional file ).

Article Snippet: Primary antibodies used were monoclonal anti nucleolin antibody (H-6, Santa Cruz), peroxidase coupled Strep-Tactin® (IBA) and polyclonal anti HDGF antibody.

Techniques: Mass Spectrometry, Purification, Isolation, Tandem Mass Spectroscopy

Journal: BMC Biochemistry

Article Title: Hepatoma-derived growth factor and nucleolin exist in the same ribonucleoprotein complex

doi: 10.1186/1471-2091-14-2

Figure Lengend Snippet: Verification of nucleolin copurification with HDGF. ( A ) Analysis of most abundant HDGFStrep-tag copurifying proteins by Coomassie stained SDS-PAGE. Proteins purified via Strep-Tactin®-MacroPrep matrices after transfection of cells with HDGFStrep-tag or untagged HDGF (Mock) were separated using SDS-PAGE and visualized by Coomassie staining. Visible proteins specific for HDGFStrep-tag sample correspond to Ku86 (86 kDa), nucleolin (110 kDa), PARP-1 (113 kDa) and the catalytic subunit of DNA-PK (460 kDa). ( B ) To confirm mass spectrometric results, lysates of HEK293 cells expressing HDGFStrep-tag fusion proteins and their eluate fractions from HDGFStrep-tag purification were examined by Western blot with a specific nucleolin antibody. Nucleolin is only present in eluates from cells expressing HDGFStrep-tag fusion proteins. ( C ) To investigate whether endogenous HDGF also interacts with nucleolin a cellular extract of untransfected HeLa cells was loaded onto a human HDGF antibody column. Non-specific proteins were washed out with 300 mM NaCl. Specifically binding proteins were eluted with 50 mM citric acid pH 3. Western blot analysis with a monoclonal anti nucleolin antibody confirmed copurification of nucleolin with endogenous HDGF.

Article Snippet: Primary antibodies used were monoclonal anti nucleolin antibody (H-6, Santa Cruz), peroxidase coupled Strep-Tactin® (IBA) and polyclonal anti HDGF antibody.

Techniques: Copurification, Staining, SDS Page, Purification, Transfection, Expressing, Western Blot, Binding Assay

Journal: BMC Biochemistry

Article Title: Hepatoma-derived growth factor and nucleolin exist in the same ribonucleoprotein complex

doi: 10.1186/1471-2091-14-2

Figure Lengend Snippet: HDGF changes intracellular distribution of nucleolin. ( A ) To examine HDGF/nucleolin complex formation in HeLa cells, cells were transfected with either wild type HDGF or a HDGF-NLS2 mutant expression vector and fixed after 48 hours with methanol. Cells transfected with wild type HDGF (HDGFwt) show predominant nuclear localization of HDGF (red, Figure panel a) as well as nucleolin (green, Figure panel c). In contrast, induction of cytoplasmic expression of HDGF-NLS2 leads to a significant increase of cytoplasmic HDGF (red, Figure panel b) as well as nucleolin (green, Figure panel d). Cells were counterstained with DAPI (blue). ( B ) HDGF positive cells were counted (n = 100) and localisation of nucleolin was analysed. 67% of HDGF-NLS2 transfected cells show cytoplasmic localisation of nucleolin in comparison to only 7% of wild type transfected cells. *p < 0.001. ( C ) Protein lysates from HDGFStrep-tag expressing HeLa cells were incubated with or without RNAse A (25 μg/ml) over night at 4°C followed by affinity purification via Strep-Tactin®-MacroPrep spin columns. Proteins from eluate fractions were subjected to Western blot analysis using a specific nucleolin antibody (upper panel) or with peroxidase coupled Strep-Tactin® to detect HDGFStrep-tag (lower panel). In samples treated with RNAse A a complete loss of interaction can be observed.

Article Snippet: Primary antibodies used were monoclonal anti nucleolin antibody (H-6, Santa Cruz), peroxidase coupled Strep-Tactin® (IBA) and polyclonal anti HDGF antibody.

Techniques: Transfection, Mutagenesis, Expressing, Plasmid Preparation, Incubation, Affinity Purification, Western Blot

Journal: Scientific Reports

Article Title: E3 ubiquitin ligase Nedd4 inhibits AP-1 activity and TNF-α production through targeting p38α for polyubiquitination and subsequent degradation

doi: 10.1038/s41598-017-04072-2

Figure Lengend Snippet: Nedd4 deficiency enhances p38α, especially p-p38αprotein levels. ( A , C ). mRNA and protein level of Nedd4 are decreased in iBMDM cells with lentiviral-based shRNA. ( A ) iBMDM cells transduced with control or shRNA targeting to Nedd4 were collected and the total RNA was extracted with TRIzol, reverse transcribed, and analyzed for Nedd4 mRNA with Q-PCR. ( B , F ). The p38, especially p-p38 protein levels were increased in Nedd4 deficient iBMDM cells stimulated for 0-60 min (above lanes) with LPS. ( B ) iBMDM cells transduced with control or shRNA targeting to Nedd4 were subjected to immunoblot analysis. ( F ) Immunoblot analysis of Nedd4 knockout (Nedd4 −/− ) iBMDM cell lines. ( C – E ). ImageJ analysis for the immunoblot of Nedd4, total p38 and p-p38 levels in iBMDM cells transduced with control or shRNA targeting to Nedd4. ( G – I ) ImageJ analysis for the immunoblot of total p38 and p-p38 levels in two Nedd4 −/− iBMDM cell lines. The results are represented from at least three independent experiments.

Article Snippet: K48-linkage specific polyubiquitin (D905) Rabbit mAb (8081 S) and K63-linkage specific polyubiquitin (D7A11) Rabbit mAb (5621 S), p-P38 MAPK (T180/Y182) (D3F9) Rabbit mAb (4511 S), and p38 MAPK (D13E1) Rabbit mAb from CST, GAPDH rabbit IgG from Proteintech, Anti-HA antibody produced in rabbit (H6908-2ML) from Sigma, and Flag-Tag mouse mAb (KM8002) from SanJian (Tianjin, China).

Techniques: shRNA, Transduction, Control, Reverse Transcription, Western Blot, Knock-Out

Journal: Scientific Reports

Article Title: E3 ubiquitin ligase Nedd4 inhibits AP-1 activity and TNF-α production through targeting p38α for polyubiquitination and subsequent degradation

doi: 10.1038/s41598-017-04072-2

Figure Lengend Snippet: p38, especially p-p38α might be involved in Nedd4 ubiquitination system. ( A ) The smear bands above 40 kDa almost disappeared in Nedd4 −/− iBMDM cells. Overexposure for immunoblot analysis of p-p38 in wild type and Nedd4 −/− iBMDM cells stimulated for 0–60 min (above lanes) with LPS treatment. ( B ) p-p38 was degraded through lysosomal and proteasomal pathways. Immunoblot analysis of p-p38 in wild-type iBMDM cells stimulated for 0–60 min (above lanes) with LPS stimulation and Chloroquine or MG132 respectively. The results are represented from at least three independent experiments. ( C , D ) ImageJ analysis for the immunoblot of total p38 ( C ) and p-p38 ( D ) levels in iBMDM cells treated with Chloroquine or MG132 respectively.

Article Snippet: K48-linkage specific polyubiquitin (D905) Rabbit mAb (8081 S) and K63-linkage specific polyubiquitin (D7A11) Rabbit mAb (5621 S), p-P38 MAPK (T180/Y182) (D3F9) Rabbit mAb (4511 S), and p38 MAPK (D13E1) Rabbit mAb from CST, GAPDH rabbit IgG from Proteintech, Anti-HA antibody produced in rabbit (H6908-2ML) from Sigma, and Flag-Tag mouse mAb (KM8002) from SanJian (Tianjin, China).

Techniques: Ubiquitin Proteomics, Western Blot

Journal: Scientific Reports

Article Title: E3 ubiquitin ligase Nedd4 inhibits AP-1 activity and TNF-α production through targeting p38α for polyubiquitination and subsequent degradation

doi: 10.1038/s41598-017-04072-2

Figure Lengend Snippet: Nedd4 inhibits TNF-α production and AP-1 activation mediated by p38α activation. ( A ) mRNA level of TNF-α was enhanced compared with control cells. Control and Nedd4-silenced iBMDM cells were stimulated with LPS for 90 min, and total RNA was extracted. After reverse transcription, TNF-α mRNA was analyzed with Q-PCR. ( B ) ELISA of TNF-α in supernatants from control and Nedd4-silenced iBMDM cells stimulated for 0–90 min (above lanes) with LPS. Data are representative of three independent experiments with similar results (mean ± s.d.) (*, significant difference). ( C ) Nedd4 inhibits MyD88-dependent transcription of gene encoding AP-1. Luciferase assay of the induction of gene encoding AP-1 in lysates of 293T cells transfected with 150 ng pGL3-AP-1 (firefly-luciferase) reporter plasmid and 3 ng pGL3-TK (renilla-luciferase) reporter, and 50 ng MyD88-expressing plasmid plus 0, 50, 100, 200 or 300 ng Nedd4-expressing plasmid and cultured for 24 h. Empty control vector pcDNA3.1(+) was added to each sample to ensure transfection of the same amount of DNA in each. Luciferase activity is normalized to renilla luciferase activity and is presented relative to basal luciferase activity. *, p < 0.05 compared with no Nedd4 group. Data are the mean ± s.d. of 4 samples in one experiment representative of similar results obtained in three independent experiments.

Article Snippet: K48-linkage specific polyubiquitin (D905) Rabbit mAb (8081 S) and K63-linkage specific polyubiquitin (D7A11) Rabbit mAb (5621 S), p-P38 MAPK (T180/Y182) (D3F9) Rabbit mAb (4511 S), and p38 MAPK (D13E1) Rabbit mAb from CST, GAPDH rabbit IgG from Proteintech, Anti-HA antibody produced in rabbit (H6908-2ML) from Sigma, and Flag-Tag mouse mAb (KM8002) from SanJian (Tianjin, China).

Techniques: Activation Assay, Control, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Luciferase, Transfection, Plasmid Preparation, Expressing, Cell Culture, Activity Assay

Journal: Scientific Reports

Article Title: E3 ubiquitin ligase Nedd4 inhibits AP-1 activity and TNF-α production through targeting p38α for polyubiquitination and subsequent degradation

doi: 10.1038/s41598-017-04072-2

Figure Lengend Snippet: Nedd4 can interact with endogenous and exogenous p38α. ( A ) Nedd4 interacts with endogenous p-p38α. iBMDMs cells were incubated with LPS at the indicated times (30 min and 60 min), lysed, immunoprecipitated with anti-Nedd4, gel separated and detected with anti-p-p38 antibody. ( B , C ) The interaction between Nedd4 and p38αV1 requires phosphorylation of p38αV1 by MKK6. HEK293 cells co-transfected with various molecules (above lanes), lysed, immunoprecipitated with anti-Nedd4, gel separated and detected with anti-Flag antibody ( B ); immunoprecipitated with anti-Flag, gel separated and detected with anti-Nedd4 antibody ( C ). ( D ) The interaction between Nedd4 and p38αV2 does not require phosphorylation of p38αV2 by MKK6. 293T cells co-transfected with various molecules (above lanes), lysed, immunoprecipitated with anti-Flag, gel separated and detected with anti-Nedd4 antibody. ( E ) Nedd4 interacts with p-p38αV1 and p38αV2 by its WW domains. These results are represented from three independent experiments.

Article Snippet: K48-linkage specific polyubiquitin (D905) Rabbit mAb (8081 S) and K63-linkage specific polyubiquitin (D7A11) Rabbit mAb (5621 S), p-P38 MAPK (T180/Y182) (D3F9) Rabbit mAb (4511 S), and p38 MAPK (D13E1) Rabbit mAb from CST, GAPDH rabbit IgG from Proteintech, Anti-HA antibody produced in rabbit (H6908-2ML) from Sigma, and Flag-Tag mouse mAb (KM8002) from SanJian (Tianjin, China).

Techniques: Incubation, Immunoprecipitation, Phospho-proteomics, Transfection

Journal: Scientific Reports

Article Title: E3 ubiquitin ligase Nedd4 inhibits AP-1 activity and TNF-α production through targeting p38α for polyubiquitination and subsequent degradation

doi: 10.1038/s41598-017-04072-2

Figure Lengend Snippet: Nedd4 mediates polyubiquitination of p38α isoforms (Left. Ubiquitination of p38αV1; Right. Ubiquitination of p38αV2). Immunoblot analysis of anti-Flag immunoprecipitates of lysates of 293T cells co-transfected with various molecules (above lanes), probed with anti-HA ( A ), anti-K48 ubiquitin ( B ), anti-K63 ubiquitin ( C ) and anti-p-p38. For whole cell lysates, p38αV1 and p38αV2 were probed with anti-Flag, MKK6 was probed with anti-HA and Nedd4 was probed with anti-Nedd4. Lane 3 just showed the signals of ubiquitinated proteins, not MKK6.

Article Snippet: K48-linkage specific polyubiquitin (D905) Rabbit mAb (8081 S) and K63-linkage specific polyubiquitin (D7A11) Rabbit mAb (5621 S), p-P38 MAPK (T180/Y182) (D3F9) Rabbit mAb (4511 S), and p38 MAPK (D13E1) Rabbit mAb from CST, GAPDH rabbit IgG from Proteintech, Anti-HA antibody produced in rabbit (H6908-2ML) from Sigma, and Flag-Tag mouse mAb (KM8002) from SanJian (Tianjin, China).

Techniques: Ubiquitin Proteomics, Western Blot, Transfection

Journal: Scientific Reports

Article Title: E3 ubiquitin ligase Nedd4 inhibits AP-1 activity and TNF-α production through targeting p38α for polyubiquitination and subsequent degradation

doi: 10.1038/s41598-017-04072-2

Figure Lengend Snippet: Analysis for polyubiquitination sites of p38α and conformation of p38αV1 and p38αV2. ( A ) Coomassie blue stained gel indicates the bands of p38αV1 and p38αV2 with polyubiquitination. 293T cells co-transfected with p38αV1-flag or p38αV2-flag, HA-Nedd4, MKK6-HA and ubiquitin-His were lysed, immunopricipitated with Ni-column and anti-Flag purification beads, gel-separated, and stained with Coomassie blue. The square area indicates the presence of ubiquinated-p38αV1 and p38αV2. ( B ) K53(K54) residue of p38α was identified as an common ubiquitination site in p38αV1 and p38αV2 by mass spectrometry. The protein sample with ubiquitinated p38αV1 and p38αV2 were recovered from the gel, enzyme digested, and subjected to mass analysis. Representative MS/MS spectra of peptides demonstrated ubiquitination at K53(K54) of p38αV1 and p38αV2. Peak matching expected y ions is labeled. ( C ) The conformational difference between p38αV1 and p38αV2. PDB documents (model No. 1p38 for p38αV1; model No. 3py3 for p38αV2) from SWISS-model, red arrows indicate the conformational difference between p38αV1 and p38αV2.

Article Snippet: K48-linkage specific polyubiquitin (D905) Rabbit mAb (8081 S) and K63-linkage specific polyubiquitin (D7A11) Rabbit mAb (5621 S), p-P38 MAPK (T180/Y182) (D3F9) Rabbit mAb (4511 S), and p38 MAPK (D13E1) Rabbit mAb from CST, GAPDH rabbit IgG from Proteintech, Anti-HA antibody produced in rabbit (H6908-2ML) from Sigma, and Flag-Tag mouse mAb (KM8002) from SanJian (Tianjin, China).

Techniques: Staining, Transfection, Ubiquitin Proteomics, Purification, Residue, Mass Spectrometry, Tandem Mass Spectroscopy, Labeling

Journal: The FASEB Journal

Article Title: O -GlcNAcylation stabilizes β-catenin through direct competition with phosphorylation at threonine 41

doi: 10.1096/fj.13-243535

Figure Lengend Snippet: Human colorectal tumor tissue exhibits higher levels of β-catenin, O-GlcNAcylation, and OGT than does healthy tissue. Colon tumor and matching tumor-adjacent normal tissues from the same patient were analyzed by Western blot for levels of β-catenin, O-GlcNAcylation, and OGT contents. Ponceau red staining of the whole extracts confirmed equal loading (40 μg proteins/lane).

Article Snippet: Mouse monoclonal anti- O -GlcNAc (RL2; VWR International, Fontenay-sous-Bois, France), mouse monoclonal anti-phosphoT41-β-catenin (C8616; Sigma-Aldrich), rabbit polyclonal anti-phospho-T41/S45-β-catenin (9565; Cell Signaling Technology, Leiden, The Netherlands), rabbit polyclonal anti-phospho-S33/S37/T41-β-catenin (9561; Cell Signaling Technology), and rabbit polyclonal anti-HA (Santa Cruz Biotechnology) were used at a final dilution of 1:1000.

Techniques: Western Blot, Staining

Journal: The FASEB Journal

Article Title: O -GlcNAcylation stabilizes β-catenin through direct competition with phosphorylation at threonine 41

doi: 10.1096/fj.13-243535

Figure Lengend Snippet: Mice fed an HCD or injected with thiamet G exhibit increased β-catenin and O-GlcNAc levels. A) Colon homogenates from mice fed an HCD or an SD for 9 wk were analyzed by Western blot for β-catenin, O-GlcNAc, and GAPDH (loading control) levels. OGTTs were performed, and blood glucose levels were determined for the HCD- and SD-fed mice by overnight food withdrawal after glucose force-feeding (differences are statistically nonsignificant). B) Colon homogenates from mice treated with the OGA inhibitor thiamet G for 2 wk were analyzed as in panel A. Anti-O-GlcNAc antibody condition was treated with free N-acetylglucosamine to show specificity. Glycemic levels were measured for the same animals with no significant differences observed. A, B) Average ± sd ratios of β-catenin/GAPDH and O-GlcNAc/GAPDH are shown. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Mouse monoclonal anti- O -GlcNAc (RL2; VWR International, Fontenay-sous-Bois, France), mouse monoclonal anti-phosphoT41-β-catenin (C8616; Sigma-Aldrich), rabbit polyclonal anti-phospho-T41/S45-β-catenin (9565; Cell Signaling Technology, Leiden, The Netherlands), rabbit polyclonal anti-phospho-S33/S37/T41-β-catenin (9561; Cell Signaling Technology), and rabbit polyclonal anti-HA (Santa Cruz Biotechnology) were used at a final dilution of 1:1000.

Techniques: Injection, Western Blot

Journal: The FASEB Journal

Article Title: O -GlcNAcylation stabilizes β-catenin through direct competition with phosphorylation at threonine 41

doi: 10.1096/fj.13-243535

Figure Lengend Snippet: O-GlcNAcylation occurs in the D box of β-catenin and reduces its ubiquitinylation. A) MCF7 cells were cotransfected with vectors encoding FLAG-β-catenin and ubiquitin-HA and treated with siOGA and MG132. β-Catenin immunoprecipitates were analyzed by Western blot with anti-FLAG and anti-HA antibodies. B) Schematic representation of the β-catenin structure. The N-terminal region contains a D box, in which phosphorylation of 4 specific residues drives the ubiquitinylation and the subsequent proteasomal degradation of β-catenin. CK1α first phosphorylates S45, and then GSK3β successively modifies T41, S37, and S33. To investigate the O-GlcNAcylation of the D box, we used a mutant form of β-catenin in which these 4 phosphorylated residues were mutated into alanine (TM). The central region of β-catenin contains 12 imperfect sequence repeats of 42 aa (the armadillo repeats) that are essential in the interaction with many partners, including cadherins, APC, and TCF/LEF. The C-terminal region displays the transactivator function required for activation of target genes. C) MCF7 cells were transfected with FLAG-β-catenin (WT), TM-FLAG-β-catenin (TM), or an empty vector (Mock). At 48 h after transfection, the cells were lysed, and the lysates were analyzed by Western blot with anti-FLAG and -actin (loading control) antibodies. D) MCF7 cells were transfected with the vectors described above. At 36 h after transfection, the cells were treated with or without 5 mM glucosamine for 16 h and lysed, and β-catenin was immunoprecipitated with an anti-FLAG antibody. Immunoprecipitates were analyzed by Western blot with anti-O-GlcNAc and anti-FLAG antibodies. Average ± sd ratios of O-GlcNAcylated-β-catenin/total β-catenin are shown. **P < 0.01.

Article Snippet: Mouse monoclonal anti- O -GlcNAc (RL2; VWR International, Fontenay-sous-Bois, France), mouse monoclonal anti-phosphoT41-β-catenin (C8616; Sigma-Aldrich), rabbit polyclonal anti-phospho-T41/S45-β-catenin (9565; Cell Signaling Technology, Leiden, The Netherlands), rabbit polyclonal anti-phospho-S33/S37/T41-β-catenin (9561; Cell Signaling Technology), and rabbit polyclonal anti-HA (Santa Cruz Biotechnology) were used at a final dilution of 1:1000.

Techniques: Western Blot, Mutagenesis, Sequencing, Activation Assay, Transfection, Plasmid Preparation, Immunoprecipitation

Journal: The FASEB Journal

Article Title: O -GlcNAcylation stabilizes β-catenin through direct competition with phosphorylation at threonine 41

doi: 10.1096/fj.13-243535

Figure Lengend Snippet: β-Catenin is O-GlcNAcylated at S23, T40, T41, and T112 in HT29 cells. A) Nano-LC-ETD MS/MS analysis of an in-gel β-catenin trypsin digest. a) Total ion current (TIC) chromatogram, m/z 300–1700. b) Reconstructed ion chromatogram (RIC) of the [M+4H]4+ ion, m/z 772.1298, corresponding to the unmodified peptide, residues 20–49 of β-catenin. c) RIC of the [M+4H]4+ ion, m/z 822.8996, corresponding to 3 differentially O-GlcNAc-modified peptides, residues 20–49 of β-catenin. d) RIC of the [M+3H]3+ ion, m/z 1068.1693, corresponding to the unmodified peptide, residues 96–124 of β-catenin. e) RIC of the [M+3H]3+ ion, m/z 1135.8624, corresponding to the O-GlcNAc modified peptide, residues 96–124 of β-catenin. NL, normalized level. B) Sequencing by ETD-MS/MS of precursor ions corresponding to the O-GlcNAc residues S23, T40, T41, and T112 in β-catenin. a) ETD-MS/MS spectrum of a tryptic O-GlcNAc-modified peptide precursor ion at m/z 822.8996 [M+4H]4+ from β-catenin. MS/MS scans were acquired for the total duration of the chromatographic peak at a retention time of 69.4 min, as shown in panel Ac. Thirty MS/MS spectra were averaged to obtain this spectrum. The peptide AAVS(GlcNAc)HWQQQSYLDSGIHSGATTTAPSLSGK (residues 20–49 of β-catenin) contained 1 site of O-GlcNAc modification at S23. The c ion series, c4 to c13 (c4 being modified), and the z2+ ion series, z172+ to z292+ (z272+ being modified), demonstrate that S23 is O-GlcNAcylated. Fragments carrying the O-GlcNAc moiety are indicated by an asterisk. b) ETD-MS/MS spectrum of a tryptic O-GlcNAc modified peptide precursor ion at m/z 822.900 [M+4H]4+ from β-catenin. MS/MS scans were acquired for the total duration of the chromatographic peak at a retention time of 66.2 min, as shown in panel Ac. Thirty MS/MS spectra were averaged to obtain this spectrum. The peptide AAVSHWQQQSYLDSGIHSGATT(GlcNAc)TAPSLSGK (residues 20–49 of β-catenin) contained 1 site of O-GlcNAc modification at T41. The z ion series, z3 to z17 (z9 being modified); the y ion series, y3 to y10 (y9 being modified); and the c2+ ion series, c172+ to c292+ (c222+ being modified) demonstrate that T41 is O-GlcNAcylated. Fragments carrying the O-GlcNAc moiety are indicated by an asterisk. c) ETD-MS/MS spectrum of a tryptic O-GlcNAc modified peptide precursor ion at m/z 822.900 [M+4H]4+ from β-catenin. MS/MS scans were acquired for the total duration of the chromatographic peak at a retention time of 68.6 min, as shown in panel Ac. Thirty MS/MS spectra were averaged to obtain this spectrum. The peptide AAVSHWQQQSYLDSGIHSGAT(GlcNAc)TTAPSLSGK (residues 20–49 of β-catenin) contained 1 site of O-GlcNAc modification at T40. The z ion series, z4 to z17 (z10 being modified), and the c2+ ion series, c172+ to c292+ (c212+ being modified), demonstrates that T40 is O-GlcNAcylated. Fragments carrying the O-GlcNAc moiety are indicated by an asterisk. d) ETD MS/MS spectrum of a tryptic O-GlcNAc modified peptide precursor ion at m/z 1135.8624 [M+3H]3+ from β-catenin. MS/MS scans were acquired for the total duration of the chromatographic peak at a retention time of 103.3 min, as shown in panel Ae. Thirty MS/MS spectra were averaged to obtain this spectrum. The peptide AAMFPETLDEGMQIPST(GlcNAc)QFDAAHPTNVQR (residues 96–124 of β-catenin) contained 1 site of O-GlcNAc modification at T112. The z ion series, z4 to z14 (z13 being modified), demonstrates that T112 is O-GlcNAcylated. Fragments carrying the O-GlcNAc moiety are indicated by an asterisk. Horizontal brackets: charge reduced ions and fragment.

Article Snippet: Mouse monoclonal anti- O -GlcNAc (RL2; VWR International, Fontenay-sous-Bois, France), mouse monoclonal anti-phosphoT41-β-catenin (C8616; Sigma-Aldrich), rabbit polyclonal anti-phospho-T41/S45-β-catenin (9565; Cell Signaling Technology, Leiden, The Netherlands), rabbit polyclonal anti-phospho-S33/S37/T41-β-catenin (9561; Cell Signaling Technology), and rabbit polyclonal anti-HA (Santa Cruz Biotechnology) were used at a final dilution of 1:1000.

Techniques: Tandem Mass Spectroscopy, Modification, Sequencing

Journal: The FASEB Journal

Article Title: O -GlcNAcylation stabilizes β-catenin through direct competition with phosphorylation at threonine 41

doi: 10.1096/fj.13-243535

Figure Lengend Snippet: O-GlcNAcylation of β-catenin D box competes with its phosphorylation. A) CD841CoN, HT29, and HCT116 cells were cultured in the presence of increasing concentrations of glucose (Glc) or with glucosamine (GlcNH2). Whole-cell lysates were probed for O-GlcNAc level and phosphorylation of β-catenin at T41 and S45. Equal amounts of immunopurified β-catenin were loaded and analyzed according to their O-GlcNAc levels by immunoblot. B) CD841CoN, HT29, and HCT116 cells were transfected with either siOGA or siCtrl and incubated with MG132. β-Catenin, phospho-β-catenin, O-GlcNAcylation, OGA, and GAPDH (loading control) levels were determined by immunoblot. C) HEK293 cells were transfected with WT FLAG-β-catenin and incubated with MG132, with or without NButGT. Cell lysates were immunopurified with an anti-FLAG antibody, and the immunoprecipitates were analyzed with a phospho-T41-dependent antibody.

Article Snippet: Mouse monoclonal anti- O -GlcNAc (RL2; VWR International, Fontenay-sous-Bois, France), mouse monoclonal anti-phosphoT41-β-catenin (C8616; Sigma-Aldrich), rabbit polyclonal anti-phospho-T41/S45-β-catenin (9565; Cell Signaling Technology, Leiden, The Netherlands), rabbit polyclonal anti-phospho-S33/S37/T41-β-catenin (9561; Cell Signaling Technology), and rabbit polyclonal anti-HA (Santa Cruz Biotechnology) were used at a final dilution of 1:1000.

Techniques: Cell Culture, Western Blot, Transfection, Incubation

Journal: The FASEB Journal

Article Title: O -GlcNAcylation stabilizes β-catenin through direct competition with phosphorylation at threonine 41

doi: 10.1096/fj.13-243535

Figure Lengend Snippet: O-GlcNAcylation at T41, but not at S23, T40, or T112, regulates β-catenin ubiquitinylation and stability. A) Schematic representation of β-catenin showing the O-GlcNAcylation sites mapped in the study. Single-point mutants and a tetramutant of β-catenin (4M: S23A/T40A/T41A/T112A) were generated. B) HEK293T cells were transfected either with a WT or mutant FLAG-β-catenin or with an empty vector (mock). At 36 h after transfection, the cells were treated with or without 1 μM MG132 for 16 h. Cell lysates were analyzed by Western blot for β-catenin using the anti-FLAG tag and anti-GAPDH antibodies (loading control). C) Cells were transfected as described in panel B. At 48 h after transfection, a subcellular fractionation was performed, and distribution of FLAG-β-catenin in each fraction (F1, cytosol; F2, membranes; F3, nucleus; and F4, cytoskeleton) was investigated by Western blot analysis. Antibodies directed against GAPDH (F1), E-cadherin (F2 and F4), H2B (F3), and CK8 (F4) were used to ensure the purity of each fraction. D) Cells were cotransfected with the different β-catenin constructs and with a ubiquitin-HA-expressing vector. At 36 h after transfection, the cells were treated with 1 μM MG132 for 16 h and lysed, and immunoprecipitation was performed with an anti-FLAG antibody. β-Catenin immunoprecipitates were analyzed by Western blot for β-catenin (anti-FLAG) and ubiquitin (anti-HA). Asterisk indicates a nonspecific band. IgG HC, immunoglobulin G heavy chain; Ub-β-cat, ubiquitinated β-catenin.

Article Snippet: Mouse monoclonal anti- O -GlcNAc (RL2; VWR International, Fontenay-sous-Bois, France), mouse monoclonal anti-phosphoT41-β-catenin (C8616; Sigma-Aldrich), rabbit polyclonal anti-phospho-T41/S45-β-catenin (9565; Cell Signaling Technology, Leiden, The Netherlands), rabbit polyclonal anti-phospho-S33/S37/T41-β-catenin (9561; Cell Signaling Technology), and rabbit polyclonal anti-HA (Santa Cruz Biotechnology) were used at a final dilution of 1:1000.

Techniques: Generated, Transfection, Mutagenesis, Plasmid Preparation, Western Blot, FLAG-tag, Fractionation, Construct, Expressing, Immunoprecipitation

Journal: The FASEB Journal

Article Title: O -GlcNAcylation stabilizes β-catenin through direct competition with phosphorylation at threonine 41

doi: 10.1096/fj.13-243535

Figure Lengend Snippet: Perturbation of O-GlcNAc level disturbs the interaction between β-catenin and α-catenin. A) MCF7 cells were treated with the potent OGT inhibitor Ac5S-GlcNAc at a concentration of 100 μM for 16 h or with DMSO as a vehicle control and with MG132 at 1 μM. The cells were lysed, and coimmunoprecipitation was performed with an anti-β-catenin antibody. Nonimmune rabbit IgG was used as a negative control. Immunoprecipitates and 1% of whole-cell lysates (input) were analyzed by Western blot with the indicated antibodies. (Note that the anti-O-GlcNAc antibody was used with and without free N-acetylglucosamine.) B) MCF7 cells were transfected with a nontarget siRNA (siCtrl) or with an OGT siRNA (siOGT). The cells were treated with 1 μM MG132 for 16 h. At 72 h after transfection, the cells were lysed, and coimmunoprecipitation and Western blot analysis were performed as in panel A, C) The same procedure as in panel B was performed, except that an OGA siRNA (siOGA) was used.

Article Snippet: Mouse monoclonal anti- O -GlcNAc (RL2; VWR International, Fontenay-sous-Bois, France), mouse monoclonal anti-phosphoT41-β-catenin (C8616; Sigma-Aldrich), rabbit polyclonal anti-phospho-T41/S45-β-catenin (9565; Cell Signaling Technology, Leiden, The Netherlands), rabbit polyclonal anti-phospho-S33/S37/T41-β-catenin (9561; Cell Signaling Technology), and rabbit polyclonal anti-HA (Santa Cruz Biotechnology) were used at a final dilution of 1:1000.

Techniques: Concentration Assay, Negative Control, Western Blot, Transfection

Journal: The FASEB Journal

Article Title: O -GlcNAcylation stabilizes β-catenin through direct competition with phosphorylation at threonine 41

doi: 10.1096/fj.13-243535

Figure Lengend Snippet: O-GlcNAcylation of β-catenin at S23, T40, T41, and T112 does not affect its interaction with α-catenin. HEK293T cells were transfected with WT or mutant FLAG-β-catenin or an empty vector (Mock). At 36 h after transfection, the cells were treated with 1 μM MG132 for 16 h. The cells were lysed, and β-catenin was immunoprecipitated with the FLAG Tag. Immunoprecipitated β-catenin and 1% of the whole-cell lysate (inputs) were analyzed by Western blot with the indicated antibodies. Densitometric values of 3 distinct experiments are represented. NS, not significant.

Article Snippet: Mouse monoclonal anti- O -GlcNAc (RL2; VWR International, Fontenay-sous-Bois, France), mouse monoclonal anti-phosphoT41-β-catenin (C8616; Sigma-Aldrich), rabbit polyclonal anti-phospho-T41/S45-β-catenin (9565; Cell Signaling Technology, Leiden, The Netherlands), rabbit polyclonal anti-phospho-S33/S37/T41-β-catenin (9561; Cell Signaling Technology), and rabbit polyclonal anti-HA (Santa Cruz Biotechnology) were used at a final dilution of 1:1000.

Techniques: Transfection, Mutagenesis, Plasmid Preparation, Immunoprecipitation, FLAG-tag, Western Blot

Journal: Scientific Reports

Article Title: Lipidomic-based investigation into the regulatory effect of Schisandrin B on palmitic acid level in non-alcoholic steatotic livers

doi: 10.1038/srep09114

Figure Lengend Snippet: (A) Expression of Nrf2 mRNA, (B) ARE-pGL6-luc reporter activity and (C) ROS levels in FFA-treated MIHA cells with or without SchB treatment (20 μM). (D) Cell viability examined by MTT assay and (E) mRNA expression level of Nrf2 in HSC-T6 cells with or without SchB treatment (40 μM). Bar charts showing the mean ± SE (n = 3 independent experiments), * p < 0.05. FFA: FFA-treated vehicle control cells; FFA+SchB: FFA-treated SchB-treated cells.

Article Snippet: Tandem repeat of ARE was inserted into the pGL6 luciferase reporter vector (ARE-pGL6-luc) (Beyotime).

Techniques: Expressing, Activity Assay, MTT Assay

Journal: Journal of Biological Chemistry

Article Title: Identification of MAPK Phosphorylation Sites and Their Role in the Localization and Activity of Hypoxia-inducible Factor-1α

doi: 10.1074/jbc.m605058200

Figure Lengend Snippet: FIGURE 2. Recombinant GST-HIF-1 is phosphorylated in vitro by p42 MAPK. A, recombinant GST-HIF-1 was incubated without (lanes 1 and 3) or with (lanes 2 and 4) 100 units of recombinant p42 MAPK kinase for 1 h at 30 °C in phosphorylation buffer without (lanes 1 and 2) or with [-32P]ATP (lanes 3 and 4) and analyzed by 10% SDS-PAGE followed by silver staining (lanes 1 and 2) or autoradiography (lanes 3 and 4). The slower migrating GST-HIF-1 band in lane 2 coincides with the radioactive band in lane 4. Numbers indicate the positions of molecular mass markers (in kDa). B, lanes 1 and 2 same as in A but analyzed by 8% SDS-PAGE followed by Western blot with anti-HIF1.

Article Snippet: SDS-PAGE and Western Blot—Proteins were resolved by 8% SDS-PAGE, and analyzed by Coomassie Blue or Western blotting using an anti-HIF-1 mouse monoclonal antibody (BD Transduction Laboratories), a rabbit polyclonal antiGFP serum generously provided by Dr. H. Boleti (Hellenic Pasteur Institute, Athens, Greece), or antibodies against p44/42 MAP kinase and phospho-p44/42 MAP kinase (Cell Signaling, Beverly, MA).

Techniques: Recombinant, In Vitro, Incubation, Phospho-proteomics, SDS Page, Silver Staining, Autoradiography, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Identification of MAPK Phosphorylation Sites and Their Role in the Localization and Activity of Hypoxia-inducible Factor-1α

doi: 10.1074/jbc.m605058200

Figure Lengend Snippet: FIGURE 3. Distinct domains of HIF-1 are targeted by p42 MAPK and another HeLa nuclear kinase. A, relative phosphorylation level of GST-HIF-1 and its deletion mutants used as substrates for phosphoryla- tion by either p42 MAPK or HeLa nuclear extracts and analyzed as shown in C and D. Values are means of three independent experiments. Schematic representation of the mutant inserts is shown on the right. B, Coomassie BluestainingfollowingSDS-PAGEoftheindicatedGST-taggedHIF-1fragments.Theamountsofrecombinant protein analyzed in the gel were also used for the phosphorylation assays shown in C and D. C and D, autora- diography following SDS-PAGE of the indicated HIF-1 forms phosphorylated by 50 units of recombinant p42 MAPK (C) or 2 g of HeLa nuclear protein extract (D) for 30 min at 30 °C. Dots indicate the position of the recombinant GST-tagged HIF-1 fragments.

Article Snippet: SDS-PAGE and Western Blot—Proteins were resolved by 8% SDS-PAGE, and analyzed by Coomassie Blue or Western blotting using an anti-HIF-1 mouse monoclonal antibody (BD Transduction Laboratories), a rabbit polyclonal antiGFP serum generously provided by Dr. H. Boleti (Hellenic Pasteur Institute, Athens, Greece), or antibodies against p44/42 MAP kinase and phospho-p44/42 MAP kinase (Cell Signaling, Beverly, MA).

Techniques: Phospho-proteomics, Mutagenesis, SDS Page, Recombinant

Journal: Journal of Biological Chemistry

Article Title: Identification of MAPK Phosphorylation Sites and Their Role in the Localization and Activity of Hypoxia-inducible Factor-1α

doi: 10.1074/jbc.m605058200

Figure Lengend Snippet: FIGURE 4. Ser-641 and Ser-643 of HIF-1 are p42 MAPK phosphorylation sites. A, MS/MS spectrum of the (M 2H)2 747.4 m/z ion, corresponding to phosphorylated ILIASPSPTHIHK peptide. Fragment 698.4 represents loss of H3PO4. B, schematic representation of HIF-1 showing the position of the peptide sequence identified as a phosphopeptide by mass spectrometry in A and alignment of corresponding amino acid sequences from vertebrate HIF-1 homo- logues. Arrows indicate the mutation of the candidate phosphoacceptor serine residues into alanines. C, GST-HIF-1 or its point mutants (2 g each) as indicated were subjected to in vitro phosphorylation by either 50 units of recombinant p42 MAPK (left panels) or 2 g of HeLa nuclear protein extracts (right panels) and analyzed by SDS-PAGE followed by Coomassie Blue staining (upper panels) or autoradiography (lower panels). D, same as in C but the nuclear protein extracts used were prepared from cells treated with 50 M PD98059 for 5 h or left untreated as indicated. Only the relevant parts of the gels are shown. The numbers under each lane represent relative phosphorylation levels measured as described under “Experimental Procedures” and are means of three independent experiments.

Article Snippet: SDS-PAGE and Western Blot—Proteins were resolved by 8% SDS-PAGE, and analyzed by Coomassie Blue or Western blotting using an anti-HIF-1 mouse monoclonal antibody (BD Transduction Laboratories), a rabbit polyclonal antiGFP serum generously provided by Dr. H. Boleti (Hellenic Pasteur Institute, Athens, Greece), or antibodies against p44/42 MAP kinase and phospho-p44/42 MAP kinase (Cell Signaling, Beverly, MA).

Techniques: Phospho-proteomics, Tandem Mass Spectroscopy, Sequencing, Mass Spectrometry, Mutagenesis, In Vitro, Recombinant, SDS Page, Staining, Autoradiography

Journal: Journal of Biological Chemistry

Article Title: Identification of MAPK Phosphorylation Sites and Their Role in the Localization and Activity of Hypoxia-inducible Factor-1α

doi: 10.1074/jbc.m605058200

Figure Lengend Snippet: FIGURE 5. Mutations in the MAPK phosphorylation sites of HIF-1 render itinactiveinHeLacells.A,Westernblotanalysisofimmunoprecipitatesfrom HeLa cells transfected with GFP alone (lane 1), wild-type GFP-HIF-1 (lanes 2 and 3), or its phosphorylation-deficient mutants (lanes 4–6) using anti-GFP antibodies for immunoprecipitation and anti-HIF-1 or anti-GFP antibodies forblottingasindicated.Panelsshowonlytherelevantpartsoftheblot:upper two panels for GFP-HIF-1; lower panel for GFP. In all cases cells were treated with MG132 and in one case (lane 3) PD98059 was also added. B, HIF-1 tran- scriptional activity determined 24 h after transfection of HeLa cells with the pGL3–5HRE-VEGF reporter plasmid, CMV-lacZ, and pEGFP-C1 plasmids carry- ing the indicated inserts. Values determined as a ratio of firefly luciferase activity over -galactosidase activity are expressed in relation to the results obtained from cells expressing GFP alone and represent the mean of four different experiments performed in triplicate (S.E.). In one case (HIF-1 PD) cells were treated with PD98059. C, Western blotting analysis of cytoplas- mic (cyt) and nuclear (nuc) extracts prepared from HeLa cells treated on not with PD98059 under the identical conditions as in B, using an antibody against MAPK (upper panel) or against its phosphorylated active form (lower panel). Only the relevant parts of the blots are shown.

Article Snippet: SDS-PAGE and Western Blot—Proteins were resolved by 8% SDS-PAGE, and analyzed by Coomassie Blue or Western blotting using an anti-HIF-1 mouse monoclonal antibody (BD Transduction Laboratories), a rabbit polyclonal antiGFP serum generously provided by Dr. H. Boleti (Hellenic Pasteur Institute, Athens, Greece), or antibodies against p44/42 MAP kinase and phospho-p44/42 MAP kinase (Cell Signaling, Beverly, MA).

Techniques: Phospho-proteomics, Transfection, Immunoprecipitation, Activity Assay, Plasmid Preparation, Luciferase, Expressing, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Identification of MAPK Phosphorylation Sites and Their Role in the Localization and Activity of Hypoxia-inducible Factor-1α

doi: 10.1074/jbc.m605058200

Figure Lengend Snippet: FIGURE 8. Treatment with LMB restores the transcriptional activity of the HIF-1 MAPK site mutants. Transcriptional activity of GFP- (A) or GAL4-DBD (B) fusion constructs of wild-type HIF-1 or its SDMA mutant determined 24 h after transfection of HeLa cells together with the corresponding reporter and control plas- mids and 4 h incubation in the absence or presence of 20 ng/ml LMB as indicated. Values (relative luciferase units, RLU), determined as a ratio of firefly over Renilla luciferase activity in abstract units, represent the mean of two independent experiments performed in triplicate (S.E.).

Article Snippet: SDS-PAGE and Western Blot—Proteins were resolved by 8% SDS-PAGE, and analyzed by Coomassie Blue or Western blotting using an anti-HIF-1 mouse monoclonal antibody (BD Transduction Laboratories), a rabbit polyclonal antiGFP serum generously provided by Dr. H. Boleti (Hellenic Pasteur Institute, Athens, Greece), or antibodies against p44/42 MAP kinase and phospho-p44/42 MAP kinase (Cell Signaling, Beverly, MA).

Techniques: Activity Assay, Construct, Mutagenesis, Transfection, Control, Incubation, Luciferase

Journal: Journal of Biological Chemistry

Article Title: Identification of MAPK Phosphorylation Sites and Their Role in the Localization and Activity of Hypoxia-inducible Factor-1α

doi: 10.1074/jbc.m605058200

Figure Lengend Snippet: FIGURE 9. A proposed model for the regulation of the subcellular distri- butionandactivationofHIF-1byaMAPK-andCRM1-dependentmech- anism. HIF-1 that enters the cell nucleus after stabilization is rapidly exported back to the cytoplasm by a CRM1-dependent mechanism. Activa- tion of the MAPK pathway causes phosphorylation of HIF-1-Ser-641/643 by p44/22 MAPK and inhibition of its export. Thus, HIF-1 accumulates insides the nucleus, heterodimerizes with ARNT, binds to HRE sequences in the pro- moter/enhancer regions of its target genes, and stimulates transcription by interacting with co-activators such as CBP/p300. This interaction can be fur- ther enhanced by MAPK-dependent phosphorylation of CBP/p300 (21).

Article Snippet: SDS-PAGE and Western Blot—Proteins were resolved by 8% SDS-PAGE, and analyzed by Coomassie Blue or Western blotting using an anti-HIF-1 mouse monoclonal antibody (BD Transduction Laboratories), a rabbit polyclonal antiGFP serum generously provided by Dr. H. Boleti (Hellenic Pasteur Institute, Athens, Greece), or antibodies against p44/42 MAP kinase and phospho-p44/42 MAP kinase (Cell Signaling, Beverly, MA).

Techniques: Phospho-proteomics, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: Cellular expression of cathepsin V with PE results in (Met)enkephalin production in PC12 cells. a, expression of cathepsin V with PE results in ME production. The human cathepsin V cDNA in expression plasmid vector was transfected into PC12 neuroendocrine cells, and measurement of ME by RIA was conducted 3 days after transfection of PE. The coexpression of cathepsin V with PE resulted in increased cellular levels of (Met)enkephalin, expressed as x ± S.E. *, statistically significant (n = 3 for each experiment, p < 0.05 by Student's t test, repeated twice). b, shown is control expression of PE alone. Transfection of the PE cDNA in PC12 cells results in expression of PE shown as a band of ∼31 kDa (lane 2). Control experiments show that PE is absent in cells when transfected with vector alone (pcDNA3.1 without PE cDNA) (lane 1). c, shown is control expression of cathepsin V alone. Transfection of the cathepsin V cDNA results in expression of this protease, shown as a main band of ∼24-kDa cathepsin V (lane 2), likely corresponding to the mature form of the enzyme of 23,999 daltons (∼24 kDa) calculated molecular mass (31). Cell extract (15 μg protein) contained ∼10–25 ng of cathepsin V, based on the high sensitivity of the Western blot with standard cathepsin V (shown in supplemental Fig. S1). The Western blot detects low levels of cathepsin V, illustrated by detection of 25 ng of purified cathepsin V. Furthermore, the anti-cathepsin V Western blot shows specificity for detection of cathepsin V but does not detect the related cysteine cathepsins L, B, and H (supplemental Fig. S1).

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: Expressing, Plasmid Preparation, Transfection, Western Blot, Purification

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: Expression of cathepsin V in human neuroblastoma SK-N-MC cells results in (Met)enkephalin production. a, cathepsin V expression mediates enkephalin production. Human cathepsin V was transfected in human SK-N-MC neuroblastoma cells, and cells were harvested 2 days later. (Met)enkephalin levels in cell extracts were measured by RIA, expressed as x ± S.E. *. statistically significant (n = 6 for each experiment, p < 0.01, Student's t test, repeated twice). b, shown is control expression of cathepsin V. Transfection of the cathepsin V cDNA results in expression of the protease, shown as an ∼24-kDa band, likely corresponding to the mature form of the enzyme. The two band areas of ∼38–40 and ∼40–45 kDa in b are consistent with preprocathepsin V and procathepsin V, respectively (8, 10, 31).

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: Expressing, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: Gene silencing of cathepsin V reduces (Met)enkephalin in human neuroblastoma SK-N-MC cells. a, gene silencing of cathepsin V by siRNA reduces (Met)enkephalin production. Human neuroblastoma SK-N-MC cells were transfected with siRNA to cathepsin V as described under “Experimental Procedures.” As controls, transfection of scrambled sequences of siRNA was conducted as well as no siRNA. One day after transfection cells were harvested, and cell levels of (Met)enkephalin were measured by radioimmunoassay. A significant decrease in (Met)enkephalin occurred after transfection of cells with siRNA to cathepsin V (p < 0.05 by Student's t test) compared with the controls of scrambled siRNA or no siRNA. b, cathepsin V protein expression is substantially reduced by siRNA gene silencing. Silencing of cathepsin V expression was assessed by Western blots after transfection of cathepsin V siRNA into SK-N-MC cells. Cathepsin V was substantially reduced with 50 nm siRNA (lane 2) and partially reduced with 25 nm siRNA (lane 3) compared with control transfection with scrambled siRNA (lane 1). These data show decreased cathepsin V expression when cathepsin V siRNA is transfected in SK-N-MC cells.

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: Transfection, RIA Assay, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: In vitro processing of PE by cathepsin V generates an intermediate present in human brain. a, in vitro processing of PE by cathepsin V is shown. PE was incubated with or without human cathepsin V for 30 min and subjected to SDS-PAGE and Western blot using anti-ME (panel i) or LE (panel ii). Results show the presence of an ∼24-kDa PE-derived band recognized by both anti-ME and anti-LE antisera. b, shown is the PE-derived ∼24-kDa intermediate in human brain cortex and hippocampus. Western blots of human brain cortex (panel i) and hippocampus (panel ii) with anti-ME and anti-LE are shown. Results show the presence of an ∼24-kDa band that is recognized by both antisera, indicating the presence of a PE-derived ∼24-kDa intermediate.

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: In Vitro, Incubation, SDS Page, Western Blot, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: In vitro processing of PE by cathepsin V via cleavage at dibasic residues generates enkephalin peptides. a, shown is the PE precursor. The PE precursor contains several copies of enkephalin-related peptides consisting of ME, LE, ME-Arg-Gly-Leu (O), and ME-Arg-Phe (H), flanked by dibasic residues (KR, KK) that are known to be processed to generate mature enkephalin. b, shown are PE-derived products. Results of recombinant human cathepsin V cleavage of recombinant PE resulted in production of a ∼24-kDa (from Fig. 4). Peptide products identified by mass spectrometry are indicated by Lys-Arg-ME consisting of the KRYGGFM sequence (with the ME sequence underlined) and C-terminal peptides consisting of the sequence RFAEALPSDEEGESYSKEVPEMEKRYGGFMRF and KRFAEALPSDEEGESYSKEVPEMEKRYGGFMRF that contain the heptapeptide ME-Arg-Phe (underlined). Peptide products result from cleavage at the N-terminal side and between dibasic residues. Mass spectrometry data of peptide products are provided in supplemental Table S1, and MS/MS spectra of identified peptide products are shown in supplemental Fig. S2.

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: In Vitro, Derivative Assay, Recombinant, Mass Spectrometry, Sequencing, Tandem Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: Cathepsin V proteolytic activity with dibasic and monobasic peptide-MCA substrates Cathepsin V was incubated with peptide-MCA, and proteolytic activity of cathepsin V was measured as μmol of fluorescent AMC/h/mg of cathepsin V. All assays were performed in quadruplicate with each substrate; replicate values varied by less than 10%. These results indicate that cathepsin V cleaves at dibasic and monobasic residues. Boc, t -butoxycarbonyl.

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: Activity Assay, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: Cathepsin V in human secretory vesicles and neuronal tissues. a, cathepsin V in isolated human secretory vesicles from sympathoadrenal pheochromocytoma. Western blot with anti-cathepsin V was assessed in soluble and membrane fractions (lanes 1 and 2, respectively) of secretory vesicles isolated from human pheochromocytoma of sympathoadrenal tissue. Blots illustrate the presence of mature cathepsin V of an ∼24-kDa band (31). b–d, shown is cathepsin V in human brain cortex and hippocampus. Cathepsin V in human neuronal tissues was analyzed by Western blots after immunoprecipitation of cathepsin V from human brain cortex (C) and hippocampus (H) (panel b, lanes 1 and 2, respectively), human SH-SY-5Y neuroblastoma cells (panel c), and human SK-N-MC neuroblastoma cells (panel d). Cathepsin V was observed as its mature form of ∼24 kDa (31). The band in panels b–d of ∼40–45 kDa are consistent to that of preprocathepsin V (8, 10, 31). The specificity of the anti-cathepsin V Western blot shows its sensitive detection of 25 ng or lower levels of standard cathepsin V, with no detection of the cysteine cathepsins L, B, or H (supplemental Fig. 1). In addition, control Western blot without primary antibody resulted in a lack of immunopositive bands.

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: Isolation, Western Blot, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: Neuronal localization of cathepsin V with enkephalin in secretory vesicles assessed by immunofluorescent confocal microscopy in human SK-N-MC neuroblastoma cells. a, localization of cathepsin V with enkephalin in secretory vesicles is shown. Cathepsin V was observed (red fluorescence) as a punctate, discrete pattern of localization like that of (Met)enkephalin (green fluorescence). The colocalization of the protease with enkephalin was illustrated by the merged yellow fluorescence. Quantitation of the relative amount of cellular cathepsin V that is colocalized with enkephalin was assessed with the Pearson correlation coefficient (Rr value) (Table 2). Measurement of the Rr value as 0.45 indicates partial colocalization of cathepsin V with enkephalin. It is noted that cathepsin V is also observed in nuclei; this is consistent with other reports demonstrating nuclear functions of cathepsin V (52, 53). b, shown is an enlarged view of secretory vesicles containing cathepsin V and enkephalin. An enlarged image more clearly shows the secretory vesicle colocalization of cathepsin V (red immunofluorescence) with enkephalin (green immunofluorescence) as indicated by the merged yellow immunofluorescence; arrows indicate the secretory vesicles containing both cathepsin V and enkephalin. Controls using normal mouse IgG instead of mouse anti-cathepsin V resulted in markedly reduced immunofluorescence (supplemental Fig. 3), illustrating the specificity of the antibody to cathepsin V.

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: Confocal Microscopy, Fluorescence, Quantitation Assay, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: Quantitation of cathepsin V localization with enkephalin and NPY in human neuroblastoma cells The colocalization of cathepsin V immunofluorescence with (Met)enkephalin in human neuroblastoma SK-N-MC cells and with NPY in human neuroblastoma SH-SY-5Y cells was quantitated by measuring the Pearson correlation coefficient (Rr) with the Velocity software as described under “Experimental Procedures.” A Pearson correlation of 1 indicates complete colocalization, and a value of 0 indicates no specific colocalization. Measurements of the Pearson correlation coefficient of 0.448 ± 0.018 for cathepsin V and (Met)enkephalin indicate their partial colocalization. The Pearson correlation coefficient of 0.736 ± 0.030 for cathepsin V and NPY in SH-SY-5Y cells indicates a reasonable degree of partial colocalization. Additional evaluation of cathepsin V localization with Lamp-1, a marker for lysosomes (54), indicated the Rr value of 0.535 ± 0.036, indicating partial colocalization. The Pearson correlation coefficient was measured with n = 5 cells. The mean ± S.E. is shown, and statistical significance of colocalization ( p < 0.0001 by Student's t test) was compared to the null hypothesis of no specific colocalization (Pearson correlation coefficient value of 0).

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: Quantitation Assay, Immunofluorescence, Software, Marker

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: Localization of cathepsin V with Lamp-1 marker of lysosomes in human SK-N-MC cells. a, shown is cathepsin V localization assessed with Lamp-1. Cathepsin V subcellular distribution was evaluated with Lamp-1, a marker for lysosomes (32). Cathepsin V (red) and Lamp-1 (green) display partial colocalization (merged yellow immunofluorescence), shown by arrows. Quantitative analyses of their partial colocalization was conducted with the Pearson correlation coefficient, indicated as 0.535 ± 0.036 (see Table 2, legend). b, shown is an enlarged view of cathepsin V localization with Lamp-1. An enlarged image shows the distinct colocalization of cathepsin V (red) with Lamp-1 (green) shown by the merged yellow immunofluorescence.

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: Marker, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: Cellular production of NPY by cathepsin V. a, localization of cathepsin V and NPY in human SH-SY-5Y neuroblastoma cells is shown. Cathepsin V localization in human SH-SY-5Y neuroblastoma cells was observed by immunofluorescent confocal microscopy. Cathepsin V (red fluorescence) and NPY (green fluorescence) were discretely localized, with colocalization among many secretory vesicles (yellow immunofluorescence, indicated by arrows). Quantitation of the relative cathepsin V colocalization with NPY was assessed by the Pearson correlation coefficient (Rr value) (Table 2). The measured Rr value of 0.74 indicates that cathepsin V is partially colocalized with NPY. Controls using normal mouse IgG instead of mouse anti-cathepsin V resulted in markedly reduced immunofluorescence (supplemental Fig. S3), illustrating the specificity of the antibody to cathepsin V. b, NPY production by cathepsin V in PC12 cells is shown. PC12 cells were cotransfected with proNPY cDNA (in pcDNA3.1 vector) and cathepsin V cDNA (in pCMV6-XL5 vector), and cells were harvested 3 days later for analyses of cellular levels of NPY (by RIA). Results are shown as x ± S.E. *, statistically significant (n = 6, p < 0.001, Student's t test).

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: Confocal Microscopy, Fluorescence, Immunofluorescence, Quantitation Assay, Plasmid Preparation

Journal: Cancer prevention research (Philadelphia, Pa.)

Article Title: Mechanistic Contribution of Ubiquitous 15-Lipoxygenase-1 Expression Loss in Cancer Cells to Terminal Cell Differentiation Evasion

doi: 10.1158/1940-6207.CAPR-10-0280

Figure Lengend Snippet: Effects of 15-LOX-1 downregulation on terminal cell differentiation, tight junction formation, and E-cadherin membrane localization in colon cells. Caco-2 wild-type (WT) cells and clones of 15-LOX-1 shRNA stably transfected Caco-2 cells with either successful [15-LOX-1 KD (+)] or unsuccessful [15-LOX-1 KD (−)] 15-LOX-1 knockdown were treated with sodium butyrate (NaBT) for 48 hours and then harvested and processed for 15-LOX-1 expression by Western blotting (Fig. 5A) and alkaline phosphatase enzymatic activity as a marker of cell differentiation (Fig. 5B). Values are the means and standard errors of the means of triplicate measurements. * P < 0.0001 (two-way analysis of variance). Positive control in A is cell lysate protein extract from HCT-116 cells transfected with a 15-LOX-1 expression vector. C, 15-LOX-1 KD (+) and 15-LOX-1 KD (−) cells were cultured for 14 days after confluence. 15-LOX-1 mRNA levels were measured by quantitative real-time reverse transcription-polymerase chain reaction on days 0 and 14 after confluence. Values are the means and standard errors of the means of triplicate measurements. * P < 0.0001 (two-way analysis of variance). D, 15-LOX-1 KD (+) and 15-LOX-1 KD (−) cells were evaluated for tight junction formation using FITC-dextran fluorescence permeability assay on day 14 after confluence. Relative fluorescence intensity values (relative to that of wild-type Caco-2 cells) at various time points are shown. Values are the means and standard errors of the means of triplicate measurements of three experiments. * P < 0.008 (t-test). E, 15-LOX-1 KD (+) and 15-LOX-1 KD (−) cells were cultured for the indicated numbers of days after confluence. Equal protein amounts from cytoskeleton-associated (insoluble) and total cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation followed by Western blotting. Band intensities (lower panel) for the blotted E-cadherin were quantified using an Odyssey fluorescent imager (LI-COR Biosciences, Lincoln, NE) and Odyssey software.

Article Snippet: We selected the pRS-15-LOX-1-shRNA-83 vector (OriGene) containing 29-base-pair shRNA forward sequence of 15-LOX-1: AGGCTTCTCTCCAGATGTCCATCACTTGG for the stable transfection experiments on the basis of preliminary studies showing efficient 15-LOX-1 gene downregulation.

Techniques: Cell Differentiation, Clone Assay, shRNA, Stable Transfection, Transfection, Expressing, Western Blot, Activity Assay, Marker, Positive Control, Plasmid Preparation, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Fluorescence, Permeability, Polyacrylamide Gel Electrophoresis, Software

Journal: Cancer prevention research (Philadelphia, Pa.)

Article Title: Mechanistic Contribution of Ubiquitous 15-Lipoxygenase-1 Expression Loss in Cancer Cells to Terminal Cell Differentiation Evasion

doi: 10.1158/1940-6207.CAPR-10-0280

Figure Lengend Snippet: 15-LOX-1 and differentiation of primary NHBE cells. Primary NHBE cells were grown for 3 weeks in an undifferentiated state in immersion cultures or in air-liquid interface cultures to induce terminal differentiation into bronchial epithelial-like structures. Cells were then harvested and processed for quantitative real-time reverse transcription-polymerase chain reaction (panel A, differentiated vs. undifferentiated NHBE cells, P = 0.0005), Western blotting (panel B), and 13-HODE levels by liquid chromatography tandem mass spectroscopy (panel C, differentiated vs. undifferentiated NHBE cells, P = 0.0002). + represents 15-LOX-1 positive control (HCT-116 colon cancer cells transfected with 15-LOX-1 expression vector).

Article Snippet: We selected the pRS-15-LOX-1-shRNA-83 vector (OriGene) containing 29-base-pair shRNA forward sequence of 15-LOX-1: AGGCTTCTCTCCAGATGTCCATCACTTGG for the stable transfection experiments on the basis of preliminary studies showing efficient 15-LOX-1 gene downregulation.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Liquid Chromatography, Tandem Mass Spectroscopy, Positive Control, Transfection, Expressing, Plasmid Preparation

Journal: Cancer prevention research (Philadelphia, Pa.)

Article Title: Mechanistic Contribution of Ubiquitous 15-Lipoxygenase-1 Expression Loss in Cancer Cells to Terminal Cell Differentiation Evasion

doi: 10.1158/1940-6207.CAPR-10-0280

Figure Lengend Snippet: 15-LOX-1 and P16 mRNA and protein expression levels in cancer cell lines. A and B, A total of 128 cancer cell lines (Supplementary Table S1) were cultured and processed for 15-LOX-1 (panel A) and p16 (panel B) mRNA by quantitative real-time reverse transcription-polymerase chain reaction. Dots in the dot plots are means of triplicate measurements from each cell line. The relative expression levels were calculated relative to expression of the calibrator sample (differentiated NHBE cells). Solid lines represent the median value for each group. C, 15-LOX-1 relative expression levels in cancer cell lines compared to the level in Caco-2 cells terminally differentiated by sodium butyrate treatment. 15-LOX-1 mRNA measurements are as in panel A but with terminally differentiated Caco-2 cells as the calibrator sample. Dots in the dot plots are means of triplicate measurements from each cell line. The solid line represents the median value for the relative expression levels. D, 15-LOX-1 protein expression in cancer cell lines. Cell lines—including cell lines with 15-LOX-1 mRNA expression levels nearly equal to or greater than the level in differentiated Caco-2 cells or NHEK—were cultured, processed for Western blotting, and probed with 15-LOX-1 antibody. Three repeated experiments yielded similar results.

Article Snippet: We selected the pRS-15-LOX-1-shRNA-83 vector (OriGene) containing 29-base-pair shRNA forward sequence of 15-LOX-1: AGGCTTCTCTCCAGATGTCCATCACTTGG for the stable transfection experiments on the basis of preliminary studies showing efficient 15-LOX-1 gene downregulation.

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Cancer prevention research (Philadelphia, Pa.)

Article Title: Mechanistic Contribution of Ubiquitous 15-Lipoxygenase-1 Expression Loss in Cancer Cells to Terminal Cell Differentiation Evasion

doi: 10.1158/1940-6207.CAPR-10-0280

Figure Lengend Snippet: 15-LOX-1 expression in normal and cancerous lung cells. A, Non-tumorigenic immortalized normal human bronchial epithelial cells (HBEC) (HBEC3KT, HBEC6KT, HBEC12KT, and HBEC24KT) and H460 lung cancer cells were grown in air-liquid interface cultures that were paraffin embedded, sectioned, and examined with hematoxylin-eosin (H&E) staining and 15-LOX-1 immunohistochemistry staining (IHC). B, Paired normal and non-small cell lung cancer tissues were immunohistochemically stained for expression of 15-LOX-1. Values are the intensity scores for 15-LOX-1 immunohistochemistry cytoplasmic staining (IHC intensity scores) in each individual case as listed in the table. NA: tissue samples were not available.

Article Snippet: We selected the pRS-15-LOX-1-shRNA-83 vector (OriGene) containing 29-base-pair shRNA forward sequence of 15-LOX-1: AGGCTTCTCTCCAGATGTCCATCACTTGG for the stable transfection experiments on the basis of preliminary studies showing efficient 15-LOX-1 gene downregulation.

Techniques: Expressing, Staining, Immunohistochemistry

Journal: Cancer prevention research (Philadelphia, Pa.)

Article Title: Mechanistic Contribution of Ubiquitous 15-Lipoxygenase-1 Expression Loss in Cancer Cells to Terminal Cell Differentiation Evasion

doi: 10.1158/1940-6207.CAPR-10-0280

Figure Lengend Snippet: Effects of 15-LOX-1 expression on Caco-2 cell spontaneous enterocyte differentiation. A and B, Effects of 15-LOX-1 shRNA on induced 15-LOX-1 expression during Caco-2 cell spontaneous enterocyte differentiation. Caco-2 cells were stably transfected with either non-targeted shRNA (control shRNA) or 15-LOX-1 shRNA and cultured for 14 days after confluence. Cells were collected at the time of confluence (0) and 14 days after confluence. 15-LOX-1 mRNA levels were measured by quantitative real-time reverse transcription-polymerase chain reaction (A) and Western blotting (B). The relative expression levels in A were calculated relative to the expression level of the calibrator sample (control shRNA, day 0). Values shown are the means ± standard deviations of triplicate measurements. * P < 0.0001. Positive control in B is cell lysate protein extract from HCT-116 cells transfected with a 15-LOX-1 expression vector. C, Effects of 15-LOX-1 on the morphology of Caco-2 cells. Caco-2 cells were cultured as described for panel A for 14 days after confluence and then fixed and examined by transmission electron microscopy. Magnifications are as indicated for each of the images. D and E, Effects of 15-LOX-1 downregulation on cell-cell contact and tight junction assembly. Caco-2 cells transfected with 15-LOX-1 shRNA or control vector were cultured to induce terminal differentiation for 14 days after confluence. Cells were processed for immunofluorescence using either ZO-1 or E-cadherin antibodies. Representative confocal microscope-captured images for staining with ZO-1 (D) and E-cadherin (E) are shown. The images for ZO-1 in the right column represent enlarged sections of the images in the left column. These enlargements demonstrate the differences in the plasma membrane structure of the cells. Bar, 10 µm.

Article Snippet: We selected the pRS-15-LOX-1-shRNA-83 vector (OriGene) containing 29-base-pair shRNA forward sequence of 15-LOX-1: AGGCTTCTCTCCAGATGTCCATCACTTGG for the stable transfection experiments on the basis of preliminary studies showing efficient 15-LOX-1 gene downregulation.

Techniques: Expressing, shRNA, Stable Transfection, Transfection, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Western Blot, Positive Control, Plasmid Preparation, Transmission Assay, Electron Microscopy, Immunofluorescence, Microscopy, Staining

Journal: Journal of Neural Transmission

Article Title: A53T-alpha-synuclein-overexpression in the mouse nigrostriatal pathway leads to early increase of 14-3-3 epsilon and late increase of GFAP

doi: 10.1007/s00702-011-0717-3

Figure Lengend Snippet: Immunohistochemical validation of upregulated striatal proteins. Striatal tissue from late age animals was analyzed. 14-3-3 epsilon staining showed neuronal immunoreactivity throughout the cytoplasm in wild-type ( a) and A53T-SNCA overexpressing mice ( b ) (corpus callosum on the left side and the ventricle below). In the mutant tissue, the neuronal cytoplasmic staining was enhanced, in agreement with the proteome results, with stronger signal intensity at the cell border (see high-magnification inset). GFAP staining showed comparable astrogliosis throughout the corpus callosum of wild-type ( c ) and mutant ( d ) aged animals (corpus callosum on the right side and the ventricle below), while the astrogliosis in the striatum appeared enhanced in the A53T-SNCA overexpressing mice, again in concordance with the proteome data

Article Snippet: After washing with 0.1% Tris–HCl, sections were incubated with biotinylated goat anti-mouse (for 14-3-3 epsilon) or anti-rabbit (for GFAP) IgG (1:200; Vector Laboratories) for 90 min at 25°C.

Techniques: Immunohistochemical staining, Staining, Mutagenesis

Journal: Journal of Neural Transmission

Article Title: A53T-alpha-synuclein-overexpression in the mouse nigrostriatal pathway leads to early increase of 14-3-3 epsilon and late increase of GFAP

doi: 10.1007/s00702-011-0717-3

Figure Lengend Snippet: Proteins identified in striata of cytosolic lysates of A53T transgenic mice by nanoLC–ESI–MS/MS analysis

Article Snippet: After washing with 0.1% Tris–HCl, sections were incubated with biotinylated goat anti-mouse (for 14-3-3 epsilon) or anti-rabbit (for GFAP) IgG (1:200; Vector Laboratories) for 90 min at 25°C.

Techniques: Transgenic Assay, Sequencing, Concentration Assay, Marker, Activity Assay, Mutagenesis

Journal: Journal of Neural Transmission

Article Title: A53T-alpha-synuclein-overexpression in the mouse nigrostriatal pathway leads to early increase of 14-3-3 epsilon and late increase of GFAP

doi: 10.1007/s00702-011-0717-3

Figure Lengend Snippet: Detailed description of the expression changes of 14-3-3 proteins. a Mass spectrum of a peptide identifying 14-3-3 epsilon. Mass spectrometric analysis of the protein spot 2 (see Fig. ) unequivocally identified the epsilon isoform of 14-3-3 protein family based on the C-terminal peptide sequence EALQDVEDENQ. b Alignments of the C-termini of several 14-3-3 protein isoforms demonstrate that the peptide identified by MS/MS ( a ) corresponds to the epsilon isoform of the 14-3-3 protein family

Article Snippet: After washing with 0.1% Tris–HCl, sections were incubated with biotinylated goat anti-mouse (for 14-3-3 epsilon) or anti-rabbit (for GFAP) IgG (1:200; Vector Laboratories) for 90 min at 25°C.

Techniques: Expressing, Sequencing, Tandem Mass Spectroscopy

Journal: Journal of Neural Transmission

Article Title: A53T-alpha-synuclein-overexpression in the mouse nigrostriatal pathway leads to early increase of 14-3-3 epsilon and late increase of GFAP

doi: 10.1007/s00702-011-0717-3

Figure Lengend Snippet: Validation of elevated 14-3-3 epsilon levels. The overexpression of 14-3-3 epsilon detected in the 2D-DIGE study was validated with independent techniques in the nigrostriatal projection via immunoblotting and qPCR analyses. a Analysis of 14-3-3 epsilon protein levels in the striatum via immunoblotting confirmed increased 14-3-3 epsilon expression. Beta-actin was used as endogenous control for normalization. b Analysis of 14-3-3 epsilon transcript levels in the midbrain via qPCR showed significantly increased 14-3-3 epsilon expression, while the levels in the striatum were not increased. TATA-binding protein ( Tbp ) was used as endogenous control. c Analysis of protein levels of the different 14-3-3 isoforms in aged striatum via immunoblotting demonstrated significantly increased 14-3-3 eta levels and significantly decreased 14-3-3 gamma levels. Data sets were normalized to the corresponding WT mean values, Student’s t test was applied for statistical analyses and significant alterations were highlighted with asterisks (* p < 0.05). N = 3–8 animals/genotype, a shows the combined data derived from two independent experiments

Article Snippet: After washing with 0.1% Tris–HCl, sections were incubated with biotinylated goat anti-mouse (for 14-3-3 epsilon) or anti-rabbit (for GFAP) IgG (1:200; Vector Laboratories) for 90 min at 25°C.

Techniques: Over Expression, Western Blot, Expressing, Binding Assay, Derivative Assay

Journal: Human molecular genetics

Article Title: Huntingtin turnover: modulation of huntingtin degradation by cAMP-dependent protein kinase A (PKA) phosphorylation of C-HEAT domain Ser2550.

doi: 10.1093/hmg/ddac165

Figure Lengend Snippet: Figure 4. PKA phosphorylates huntingtin at Ser2550. (A) Bar graph showing MS/MS results of each phosphopeptide from Q23-huntingtin (HTT) purified from Sf9 insect cells incubated without (open bar) or with purified PRKACA (black bar). (B) The multiple sequence alignment compares the human huntingtin amino acid sequence focused on the region of Ser2550 (Red bold) with huntingtins predicted from the gene sequence of other orthologs, Macaca mulatta, Canis lupus familiaris, Mus musculus and Danio rerio. Shading denotes the conservation of PKA substrate motif (RKXS) across these organisms. (C) Immunoblot showing the result of detecting pSer2550 in FLAG-tagged huntingtin overexpressed without or with PRKACA overexpression in HTT null HEK293T cells for 24 h, with abHTT-pS2550, as well as huntingtin level detected with anti-huntingtin reagent (MAB2166), and PRKACA level detected with anti-PRKACA reagent, and α-tubulin as loading control with anti-α-tubulin reagent. (D) Plot showing relative abundance of pSer2550 endogenous huntingtin peptide detected by PRM assay with or without overexpression of PRKACA for 24 h in HEK293T cells. Endogenous huntingtin was enriched by immunoprecipitation with MAB2166 antibody before PRM assay.

Article Snippet: The recombinant human PRKACA cDNA was obtained from Addgene (Plasmid #23495) and cloned into between BamHI and D ow nloaded from https://academ ic.oup.com /hm g/article/32/1/30/6652516 by U niversidade Federal de Santa C atarina user on 07 February 2024 KpnI in pcDNA5 vector (Invitrogen).

Techniques: Tandem Mass Spectroscopy, Phospho-proteomics, Purification, Incubation, Sequencing, Western Blot, Over Expression, Control, Immunoprecipitation

Journal: Human molecular genetics

Article Title: Huntingtin turnover: modulation of huntingtin degradation by cAMP-dependent protein kinase A (PKA) phosphorylation of C-HEAT domain Ser2550.

doi: 10.1093/hmg/ddac165

Figure Lengend Snippet: Figure 5. PKA Ser2550 phosphorylation increases the rate of huntingtin degradation. (A) The immunoblot (left) shows the results of detecting Q23- huntingtin (HTT) or Q23-huntingtin phosphomutant S2550A overexpressed in HTT null HEK293T cells in the absence or presence of PRKACA for 24 h or 48 h, with anti-huntingtin reagent (MAB2166) and PRKACA level detected with anti-PRKACA reagent, and α-tubulin as loading control with anti-α-tubulin reagent. The histogram (right) shows the huntingtin/α-tubulin band intensities normalized to the mean ratio of each sample at post 24 h transfection. Data represent mean ± s.e.m (n = 2). (B and C) Immunoblots (left) showing cycloheximide (CHX) chase experiments of normal Q23-huntingtin (Q23 HTT) (B) and mutant Q78-huntingtin (Q78 HTT) (C) overexpressed without or with PRKACA overexpression in HTT null HEK293T cells, probed with anti- huntingtin reagent (MAB2166), anti-PRKACA reagent and anti-α-tubulin reagent for huntingtin, PRKACA and α-tubulin as loading control, respectively. For each, the plots (right) show the huntingtin/α-tubulin band intensities normalized to the mean ratio of each sample at post 24-h transfection (P0) in the absence (open symbol) and presence (closed symbol) of PRKACA overexpression. Data represent mean ± s.e.m (n = 2). Asterisks indicate level of statistical significance (paired Student’s t-test, two tailed); ∗P < 0.05.

Article Snippet: The recombinant human PRKACA cDNA was obtained from Addgene (Plasmid #23495) and cloned into between BamHI and D ow nloaded from https://academ ic.oup.com /hm g/article/32/1/30/6652516 by U niversidade Federal de Santa C atarina user on 07 February 2024 KpnI in pcDNA5 vector (Invitrogen).

Techniques: Phospho-proteomics, Western Blot, Control, Transfection, Mutagenesis, Over Expression, Two Tailed Test

Journal: Human molecular genetics

Article Title: Huntingtin turnover: modulation of huntingtin degradation by cAMP-dependent protein kinase A (PKA) phosphorylation of C-HEAT domain Ser2550.

doi: 10.1093/hmg/ddac165

Figure Lengend Snippet: Figure 6. Cellular PKA activity is associated with endogenous huntingtin level. (A) The immunoblot (left) shows the expression levels of endogenous huntingtin (HTT) without or with overexpressed PRKACA in HEK293 cells, detected with anti-huntingtin reagent (MAB2166) and PRKACA levels detected with anti-PRKACA reagent and α-tubulin as loading control with anti-α-tubulin reagent. The histogram (right) shows the huntingtin/α-tubulin band intensities normalized to the mean ratio of each sample of empty vector for empty vector (open bar) and PRKACA (closed bar) expressed samples. Data represent mean ± s.e.m (n = 3). (B) The immunoblot (left) shows the expression level of endogenous huntingtin in HEK293T cells stably expressing scramble- or PRKACA-shRNAs, detected with anti-huntingtin reagent (MAB2166) and PRKACA levels detected with anti-PRKACA reagent and α-tubulin as loading control with anti-α-tubulin reagent. The histogram (right) shows the huntingtin/α-tubulin band intensities normalized to the mean ratio of each sample expressing scramble-shRNA for scramble-shRNA (open bar) and PRKACA-shRNA (closed bar) expressed samples. Data represent mean ± s.e.m (n = 3). (C) The immunoblot shows the expression level of endogenous huntingtin in hNPC.10 (Q62/Q20) treated with DMSO or 300 μM of 8-Br-cAMP, detected with anti-huntingtin reagent (MAB2166) and phosphor-CREB with anti-phosphor-CREB (S133) reagent, total CREB with anti-CREB reagent and α-tubulin as loading control with anti-α-tubulin reagent. Of note, Q62-huntingtin (red arrow) separated from Q20-huntingtin (black arrow) was also confirmed by probing with 1F8 antibody [data not shown (61)]. A histogram showing total huntingtin/α-tubulin band intensities normalized to the mean ratio of each sample at 0 h for DMSO (open symbol) and 8-Br-cAMP- (closed symbol) treated samples, respectively. Data represent mean ± s.e.m (n = 2). Asterisks indicate level of statistical significance (paired Student’s t-test, two tailed); ∗P < 0.05.

Article Snippet: The recombinant human PRKACA cDNA was obtained from Addgene (Plasmid #23495) and cloned into between BamHI and D ow nloaded from https://academ ic.oup.com /hm g/article/32/1/30/6652516 by U niversidade Federal de Santa C atarina user on 07 February 2024 KpnI in pcDNA5 vector (Invitrogen).

Techniques: Activity Assay, Western Blot, Expressing, Control, Plasmid Preparation, Stable Transfection, shRNA, Two Tailed Test

Journal: Human molecular genetics

Article Title: Huntingtin turnover: modulation of huntingtin degradation by cAMP-dependent protein kinase A (PKA) phosphorylation of C-HEAT domain Ser2550.

doi: 10.1093/hmg/ddac165

Figure Lengend Snippet: Figure 7. Inverse correlation between levels of huntingtin and PRKACA in a panel of HD neural progenitor cells. The scatter plot of the endogenous huntingtin ratio (normalized to pooled reference sample) (X-axis) versus endogenous PRKACA ratio (normalized to pooled reference sample) (Y- axis) measured by quantitative LC–MS/MS using TMT 10-plex across each of the 11 members of an hNPC panel shows an inverse correla- tion between huntingtin and PRKACA levels, as indicated by the linear regression line (black line), that is significant (Multiple R-squared: 0.443, Adjusted R-squared: 0.3812; F-statistic: 7.159 on 1 and 9 DF, P-value: 0.02539.)

Article Snippet: The recombinant human PRKACA cDNA was obtained from Addgene (Plasmid #23495) and cloned into between BamHI and D ow nloaded from https://academ ic.oup.com /hm g/article/32/1/30/6652516 by U niversidade Federal de Santa C atarina user on 07 February 2024 KpnI in pcDNA5 vector (Invitrogen).

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: Human molecular genetics

Article Title: Huntingtin turnover: modulation of huntingtin degradation by cAMP-dependent protein kinase A (PKA) phosphorylation of C-HEAT domain Ser2550.

doi: 10.1093/hmg/ddac165

Figure Lengend Snippet: Figure 4. PKA phosphorylates huntingtin at Ser2550. (A) Bar graph showing MS/MS results of each phosphopeptide from Q23-huntingtin (HTT) purified from Sf9 insect cells incubated without (open bar) or with purified PRKACA (black bar). (B) The multiple sequence alignment compares the human huntingtin amino acid sequence focused on the region of Ser2550 (Red bold) with huntingtins predicted from the gene sequence of other orthologs, Macaca mulatta, Canis lupus familiaris, Mus musculus and Danio rerio. Shading denotes the conservation of PKA substrate motif (RKXS) across these organisms. (C) Immunoblot showing the result of detecting pSer2550 in FLAG-tagged huntingtin overexpressed without or with PRKACA overexpression in HTT null HEK293T cells for 24 h, with abHTT-pS2550, as well as huntingtin level detected with anti-huntingtin reagent (MAB2166), and PRKACA level detected with anti-PRKACA reagent, and α-tubulin as loading control with anti-α-tubulin reagent. (D) Plot showing relative abundance of pSer2550 endogenous huntingtin peptide detected by PRM assay with or without overexpression of PRKACA for 24 h in HEK293T cells. Endogenous huntingtin was enriched by immunoprecipitation with MAB2166 antibody before PRM assay.

Article Snippet: Primary antibodies used in this research are as follows: mouse monoclonal anti-huntingtin antibody MAB2166 (MilliporeSigma), MAB2168 (MilliporeSigma), 1F8 (61), rabbit polyclonal anti-phospho Serine 2550 antibody (abHTT-pS2550) (20), rabbit polyclonal anti-GAPDH antibody (Santa Cruz Biotechnology), mouse monoclonal anti-α-tubulin antibody (Cell Signaling Technology), mouse monoclonal anti-PRKACA antibody (Santa Cruz Biotechnology), mouse monoclonal anti-CREB antibody (Cell Signaling Technology), rabbit monoclonal antiphospho CREB (S133) antibody (Cell Signaling Technology).

Techniques: Tandem Mass Spectroscopy, Phospho-proteomics, Purification, Incubation, Sequencing, Western Blot, Over Expression, Control, Immunoprecipitation

Journal: Human molecular genetics

Article Title: Huntingtin turnover: modulation of huntingtin degradation by cAMP-dependent protein kinase A (PKA) phosphorylation of C-HEAT domain Ser2550.

doi: 10.1093/hmg/ddac165

Figure Lengend Snippet: Figure 5. PKA Ser2550 phosphorylation increases the rate of huntingtin degradation. (A) The immunoblot (left) shows the results of detecting Q23- huntingtin (HTT) or Q23-huntingtin phosphomutant S2550A overexpressed in HTT null HEK293T cells in the absence or presence of PRKACA for 24 h or 48 h, with anti-huntingtin reagent (MAB2166) and PRKACA level detected with anti-PRKACA reagent, and α-tubulin as loading control with anti-α-tubulin reagent. The histogram (right) shows the huntingtin/α-tubulin band intensities normalized to the mean ratio of each sample at post 24 h transfection. Data represent mean ± s.e.m (n = 2). (B and C) Immunoblots (left) showing cycloheximide (CHX) chase experiments of normal Q23-huntingtin (Q23 HTT) (B) and mutant Q78-huntingtin (Q78 HTT) (C) overexpressed without or with PRKACA overexpression in HTT null HEK293T cells, probed with anti- huntingtin reagent (MAB2166), anti-PRKACA reagent and anti-α-tubulin reagent for huntingtin, PRKACA and α-tubulin as loading control, respectively. For each, the plots (right) show the huntingtin/α-tubulin band intensities normalized to the mean ratio of each sample at post 24-h transfection (P0) in the absence (open symbol) and presence (closed symbol) of PRKACA overexpression. Data represent mean ± s.e.m (n = 2). Asterisks indicate level of statistical significance (paired Student’s t-test, two tailed); ∗P < 0.05.

Article Snippet: Primary antibodies used in this research are as follows: mouse monoclonal anti-huntingtin antibody MAB2166 (MilliporeSigma), MAB2168 (MilliporeSigma), 1F8 (61), rabbit polyclonal anti-phospho Serine 2550 antibody (abHTT-pS2550) (20), rabbit polyclonal anti-GAPDH antibody (Santa Cruz Biotechnology), mouse monoclonal anti-α-tubulin antibody (Cell Signaling Technology), mouse monoclonal anti-PRKACA antibody (Santa Cruz Biotechnology), mouse monoclonal anti-CREB antibody (Cell Signaling Technology), rabbit monoclonal antiphospho CREB (S133) antibody (Cell Signaling Technology).

Techniques: Phospho-proteomics, Western Blot, Control, Transfection, Mutagenesis, Over Expression, Two Tailed Test

Journal: Human molecular genetics

Article Title: Huntingtin turnover: modulation of huntingtin degradation by cAMP-dependent protein kinase A (PKA) phosphorylation of C-HEAT domain Ser2550.

doi: 10.1093/hmg/ddac165

Figure Lengend Snippet: Figure 6. Cellular PKA activity is associated with endogenous huntingtin level. (A) The immunoblot (left) shows the expression levels of endogenous huntingtin (HTT) without or with overexpressed PRKACA in HEK293 cells, detected with anti-huntingtin reagent (MAB2166) and PRKACA levels detected with anti-PRKACA reagent and α-tubulin as loading control with anti-α-tubulin reagent. The histogram (right) shows the huntingtin/α-tubulin band intensities normalized to the mean ratio of each sample of empty vector for empty vector (open bar) and PRKACA (closed bar) expressed samples. Data represent mean ± s.e.m (n = 3). (B) The immunoblot (left) shows the expression level of endogenous huntingtin in HEK293T cells stably expressing scramble- or PRKACA-shRNAs, detected with anti-huntingtin reagent (MAB2166) and PRKACA levels detected with anti-PRKACA reagent and α-tubulin as loading control with anti-α-tubulin reagent. The histogram (right) shows the huntingtin/α-tubulin band intensities normalized to the mean ratio of each sample expressing scramble-shRNA for scramble-shRNA (open bar) and PRKACA-shRNA (closed bar) expressed samples. Data represent mean ± s.e.m (n = 3). (C) The immunoblot shows the expression level of endogenous huntingtin in hNPC.10 (Q62/Q20) treated with DMSO or 300 μM of 8-Br-cAMP, detected with anti-huntingtin reagent (MAB2166) and phosphor-CREB with anti-phosphor-CREB (S133) reagent, total CREB with anti-CREB reagent and α-tubulin as loading control with anti-α-tubulin reagent. Of note, Q62-huntingtin (red arrow) separated from Q20-huntingtin (black arrow) was also confirmed by probing with 1F8 antibody [data not shown (61)]. A histogram showing total huntingtin/α-tubulin band intensities normalized to the mean ratio of each sample at 0 h for DMSO (open symbol) and 8-Br-cAMP- (closed symbol) treated samples, respectively. Data represent mean ± s.e.m (n = 2). Asterisks indicate level of statistical significance (paired Student’s t-test, two tailed); ∗P < 0.05.

Article Snippet: Primary antibodies used in this research are as follows: mouse monoclonal anti-huntingtin antibody MAB2166 (MilliporeSigma), MAB2168 (MilliporeSigma), 1F8 (61), rabbit polyclonal anti-phospho Serine 2550 antibody (abHTT-pS2550) (20), rabbit polyclonal anti-GAPDH antibody (Santa Cruz Biotechnology), mouse monoclonal anti-α-tubulin antibody (Cell Signaling Technology), mouse monoclonal anti-PRKACA antibody (Santa Cruz Biotechnology), mouse monoclonal anti-CREB antibody (Cell Signaling Technology), rabbit monoclonal antiphospho CREB (S133) antibody (Cell Signaling Technology).

Techniques: Activity Assay, Western Blot, Expressing, Control, Plasmid Preparation, Stable Transfection, shRNA, Two Tailed Test