Journal: Spinal cord

Article Title: Sodium hyaluronate-CNTF gelatinous particles promote axonal growth, neurogenesis and functional recovery after spinal cord injury.

doi: 10.1038/sc.2014.54

Figure Lengend Snippet: Figure 4 The b-tubulin-III–positive neuron-like cells and the differentiation of endogenous progenitors in the sodium hyaluronate-CNTF group. (a) At 1 month PO in the sodium hyaluronate-CNTF group, many small and process-extending b-tubulin-III–positive neuron-like cells (shown by white arrows) were observed at the lesion edge of the host cord. (b) The b-tubulin-III–positive neuron-like cells were immunoreactive for NeuN (shown by white arrows). (c) At 1 month PO in the sodium hyaluronate-CNTF group, the b-tubulin-III–positive neuron-like cells (shown by white arrows) were detectable in the lesion area. (d) High magnification of the boxed area in panel (c). The b-tubulin-III–positive neuronal fiber (shown by white arrowhead) extended and the b-tubulin-III– positive neuron-like cell seemed to form cell contacts (shown by white arrow). (e–h) At 4 days PO in the sodium hyaluronate-CNTF group, BrdU (red) and DCX (green) double-positive cells were observed at the lesion edge of the host cord, with neuron-like appearance. The cells directed by white arrows in panel (g) are shown in panel (h). (i–l) At 11 days PO in the sodium hyaluronate-CNTF group, BrdU (red) and b-tubulin-III (green) double-positive cells were observed in the lesion area. The cell directed by the white arrow in panel (k) is shown in panel (l). The dotted lines indicate the boundary between the host and lesion area. Scale bars: (a, e–g), 100mm; (b, c and h), 50mm; d, 25 mm; (i–k), 75 mm. A full color version of this figure is available at the Spinal Cord journal online.

Article Snippet: Spinal cord sections were incubated in the following primary antibodies (Ab) at 4 1C for 48 h: mouse anti-neurofilament (Pan) monoclonal Ab (NF, 1:50, Zymed Laboratories, South San Francisco, CA, USA); rabbit anti-b-tubulin-III protein polyclonal Ab (btubulin-III, 1:100, Sigma); rabbit anti-glial fibrillary acidic protein polyclonal Ab (1:150, Zymed Laboratories); mouse anti-BrdU monoclonal Ab (1:70, Bioss, Woburn, MA, USA); and rabbit anti-doublecortin protein polyclonal Ab (DCX, 1:100, Abcam, Cambridge, MA, USA).

Techniques:

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Transcriptomics-Based Liquid Biopsy for Early Detection of Recurrence in Locally Advanced Gastric Cancer.

doi: 10.1002/advs.202406276

Figure Lengend Snippet: Figure 2. Discovery process and preliminary validation of candidate markers for postoperative recurrence in LAGC patients based on public databases and transcriptomics sequencing data. A) Four candidate mRNAs (AGTR1, DNER, EPHA7, SUSD5) were identified through a Venn diagram analysis using the TCGA database (28 recurrent patients versus 159 non-recurrent patients), transcriptome data from the GEO database (125 recurrent patients versus 157 non-recurrent patients), and paired mRNA sequencing (3 recurrent patients versus 3 non-recurrent patients). B) A volcano plot illustrates the expression levels of these four genes in recurrent and non-recurrent cancer tissues. C) The expression levels of the four candidate mRNAs (AGTR1,

Article Snippet: Membranes were probed with antibodies against AGTR1 (1:1000 dilutions, 25343-1-AP, Proteintech, Chicago, USA), DNER (1:1000 dilutions, 24362-1-AP, Proteintech, Chicago, USA), EPHA7 (1:1000 dilutions, 66667-1-Ig, Proteintech, Chicago, USA), SUSD5 (1:500 dilutions, bs-7331R, Bioss, Beijing, China), CREB (1:500 dilutions, 381013, Zenbio, Chengdu, China), p-CREB (1:500 dilutions, 380697, Zenbio, Chengdu, China), and GAPDH (1:10000 dilutions, 10494-1-AP, Proteintech, Chicago, USA).

Techniques: Biomarker Discovery, Sequencing, Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Transcriptomics-Based Liquid Biopsy for Early Detection of Recurrence in Locally Advanced Gastric Cancer.

doi: 10.1002/advs.202406276

Figure Lengend Snippet: Figure 7. Four recurrence-related mRNA genes promote GC cell proliferation, migration and invasion in vitro. A,B) Scratch assay to evaluate the migration ability of GC cells after knockdown of AGTR1 and DNER, respectively. C,D) Transwell assay to assess the invasion and metastasis abilities of GC cells after knockdown of AGTR1 and DNER, respectively. E,F) EdU assay to determine the proliferation ability of GC cells after knockdown of AGTR1 and DNER, respectively. G–J) Colony formation assay to measure the proliferation ability of GC cells after knockdown of AGTR1, DNER, EPHA7, and SUSD5. K–N) CCK-8 assay to detect the proliferation ability of GC cells after knockdown of AGTR1, DNER, EPHA7, and SUSD5. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Membranes were probed with antibodies against AGTR1 (1:1000 dilutions, 25343-1-AP, Proteintech, Chicago, USA), DNER (1:1000 dilutions, 24362-1-AP, Proteintech, Chicago, USA), EPHA7 (1:1000 dilutions, 66667-1-Ig, Proteintech, Chicago, USA), SUSD5 (1:500 dilutions, bs-7331R, Bioss, Beijing, China), CREB (1:500 dilutions, 381013, Zenbio, Chengdu, China), p-CREB (1:500 dilutions, 380697, Zenbio, Chengdu, China), and GAPDH (1:10000 dilutions, 10494-1-AP, Proteintech, Chicago, USA).

Techniques: Migration, In Vitro, Wound Healing Assay, Knockdown, Transwell Assay, EdU Assay, Colony Assay, CCK-8 Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Transcriptomics-Based Liquid Biopsy for Early Detection of Recurrence in Locally Advanced Gastric Cancer.

doi: 10.1002/advs.202406276

Figure Lengend Snippet: Figure 8. Four recurrence-related mRNA genes promote GC cell xenograft tumor growth and metastasis in vivo. A–D) Morphological images showing reduced subcutaneous xenograft tumor formation in mice injected with AGS cells knocked down for AGTR1, DNER, EPHA7, and SUSD5, along with tumor volume growth curves and final tumor weights. E) Representative IHC images of subcutaneous xenograft tumors after knockdown of AGTR1 (left), and quantification of IHC staining data for Ki67, N-cadherin, E-cadherin, and Vimentin in each group of mice (right). F) Representative IHC images of peritoneal metastasis tumors after intraperitoneal injection of AGS cells knocked down for AGTR1 (left), and quantification of IHC staining data for MMP9, N-cadherin, E-cadherin, and Vimentin in each group of mice (right). G) Representative images of peritoneal metastasis tumors in the abdominal cavity of mice injected with AGS cells knocked down for AGTR1. H) Measurement and quantification of the number of peritoneal metastatic tumors in

Article Snippet: Membranes were probed with antibodies against AGTR1 (1:1000 dilutions, 25343-1-AP, Proteintech, Chicago, USA), DNER (1:1000 dilutions, 24362-1-AP, Proteintech, Chicago, USA), EPHA7 (1:1000 dilutions, 66667-1-Ig, Proteintech, Chicago, USA), SUSD5 (1:500 dilutions, bs-7331R, Bioss, Beijing, China), CREB (1:500 dilutions, 381013, Zenbio, Chengdu, China), p-CREB (1:500 dilutions, 380697, Zenbio, Chengdu, China), and GAPDH (1:10000 dilutions, 10494-1-AP, Proteintech, Chicago, USA).

Techniques: In Vivo, Injection, Knockdown, Immunohistochemistry

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 3. Generation of Tg-MPP1 mice. A, Upper panel: scheme of the plasmid used for the generation of Tg-MPP1 mice. Lower panel: PCR genotyping of ear- punch biopsies from 11 Tg-MPP1-positive mice with stable integration of the transgenic MPP1 cDNA into the genomic DNA. The negative control (-) did not contain genomic DNA, and the linearized MPP1 plasmid DNA (P) was used as a positive control. The lane marked with M, is the DNA marker. B, Immunoblot detection of the MPP1 protein in heart protein extracts from Tg-MPP1 mice and non-transgenic B6 mice. The left panel is a representative immunoblot, and the right panel shows quantitative data (mean ± s.d., n = 4 mice per group). The p- value is indicated and was determined by the unpaired, two-tailed, t-test. The lower panel is a control immunoblot detecting α-tubulin. C, As a specificity control of the monoclonal anti-MPP1 antibody, immunoblot detection of MPP1 in MPP1-transfected HEK cells was performed in comparison to mock- transfected HEK cells. The lower blot shows a loading control detecting GAPDH.

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Plasmid Preparation, Transgenic Assay, Negative Control, Positive Control, Marker, Western Blot, Two Tailed Test, Control, Transfection, Comparison

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 2. Upregulation of the MAGUK family protein, MPP1, in three different heart failure models. A,B, Probe set intensities of cardiac Mpp iso forms were determined by whole genome microarray gene expression profiling of the AAC-induced heart failure model in comparison to sham-operated con trols (A), and of Apoe−/− mice with long-term atherosclerosis-induced heart failure in comparison to age-matched non-transgenic B6 mice (B). Affymetrix IDs of probe sets detecting Mpp1, Mpp2, Mpp3, Mpp4, Mpp5, Mpp6, and Mpp7 are indicated. Data are mean values ± s.d. (four hearts per microarray chip with two microarray chips per group). Probe set intensities are taken from NCBI GEO dataset GSE25765. C, Cardiac transcript levels of Mpp isoforms in 8-month-old, male Tg-RKIP mice were determined by NGS in comparison to age- and sex- matched, non-transgenic FVB controls (NCBI GEO dataset GSE191316) (mean ± s.d., n = 3 mice per group). Statistically significant differences between transcript levels of the heart failure groups and the respective control group were determined by Tukey’s test, and are indicated for each individual MAGUK gene (A,B,C). P-values for statistically different MAGUK genes are indicated. All other MAGUK genes were not significantly different (n.s.) between the heart failure and control groups.

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Microarray, Gene Expression, Comparison, Transgenic Assay, Control

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 4. Tg-MPP1 mice develop features of heart failure with cardiac enlarge ment at an age of 8 months. A, Echo cardiographic measurement of the left ventricular ejection fraction (LVEF, %), the fractional shortening (FS, %), the left ventricular internal diameter in diastole (LVIDd), and the left ventricular internal diameter in systole (LVIDs) of 8-month- old, male Tg-MPP1 mice, and sex- and age-matched, non-transgenic B6 mice. Echocardiographic measurements were performed under anesthesia. B, Determi nation of the body weights (BW), heart weights (HW), and the heart weight to body weight ratios (HW/BW) of 8-month- old, male Tg-MPP1 mice, and of sex- and age-matched, non-transgenic B6 mice. Data (A,B) are the mean ± s.d., n = 6 mice per group. P-values were determined by the unpaired, two-tailed t-test. C, Immu nohistological detection of MPP1 on heart sections of Tg-MPP1 mice in comparison to those of non-transgenic B6 mice (n = 4 mice/group; bar: 2 mm). Sections were stained with the anti-MPP1 antibody (MPP1) and counterstained with hema toxylin (HE). The right panels show higher magnification images of representative sections from a Tg-MPP1 mouse and a non- transgenic B6 control (bar: 20 μm).

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Transgenic Assay, Two Tailed Test, Comparison, Staining, Control

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 5. Co-localization of AGTR1 with MPP1 in vivo, and increased cardiac AGTR1 protein levels in Tg- MPP1 mice. A, Immunofluorescence detection of MPP1 and AGTR1 on cardiac cryosections from Tg-CMV- AGTR1-Cerulean mice shows co-localization of AGTR1 with MPP1 on sarcolemmal membranes (yellow). MPP1 was stained with mouse monoclonal anti-MPP1 antibody (red), AGTR1-Cerulean was stained with rabbit poly clonal anti-GFP antibodies (green), and nuclei were stained with DAPI (blue). The immunofluorescence co- localization study shows cryosections from four different mice (bar: 40 μm). B, Cardiac AGTR1-specific binding sites were determined on sarcolemmal mem branes of Tg-MPP1 mice and non-transgenic B6 mice by radioligand binding with Sar1,[125I]Tyr4,Ile8-angiotensin II. Data are shown as mean values ± s.d., n = 6 mice per group. The p-value was determined by the unpaired, two- tailed t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: In Vivo, Immunofluorescence, Staining, Binding Assay, Transgenic Assay, Two Tailed Test

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 6. MPP1 increased the cellular contents of AGTR1eYFP in HEK cells. A,B, Cellular AGTR1eYFP levels were increased by co-transfection of HEK293 cells with an MPP1-encoding pcDNA3 expression plasmid (+). Control cells were transfected with the pcDNA3 plasmid without insert (-). Panel (A) shows cellular AGTR1eYFP fluorescence peak intensities at an emission wavelength of 527 nm, and panel (B) shows representative AGTR1eYFP fluorescence emis sion spectra without (grey) and with MPP1-encoding plasmid co-transfection (red). The black line shows a spectrum of control cells transfected with pcDNA3 without insert (Cont.). C,D, Co-transfection of the MPP1-encoding plasmid did not significantly alter cellular ADRB1eYFP levels. Control cells were trans fected with the pcDNA3 plasmid without insert (-). Panel (C) shows cellular ADRB1eYFP fluorescence peak intensities at an emission wavelength of 527 nm, and panel (D) shows representative fluorescence emission spectra of ADRB1eYFP-expressing cells without and with MPP1-encoding plasmid co- transfection. Data (A,C) show mean values ± s.d. (n = 8 biological replicates). P-values were determined by Tukey’s test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Cotransfection, Expressing, Plasmid Preparation, Control, Transfection, Fluorescence

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 7. AGTR1-(1–319)-eYFP with deletion of the carboxyl terminal tail is also enhanced by MPP1 in HEK cells. A, Cellular fluorescence peak intensities at an emission wavelength of 527 nm were deter mined of HEK cells with expression of the full-length AGTR1-(1–359)-eYFP without (-) and with (+) co- transfection of the MPP1-encoding plasmid, and of HEK cells with expression of the truncated AGTR1- (1–319)-eYFP without (-), and with (+) co- transfection of MPP1. Data are mean values ± s.d. (n = 10 biological replicates). P-values were deter mined by Tukey’s test. B, Topological scheme of the full-length AGTR1-(1–359) protein sequence. Trun cated residues of AGTR1-(1–319) are marked in red. The AGTR1 topology was derived from Uniprot (P30556 AGTR1_Human), and the scheme was drawn with Protter, version 1.0. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Fluorescence, Expressing, Cotransfection, Plasmid Preparation, Sequencing, Derivative Assay

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 9. The AGTR1-enhancing effect mediated by MPP1 requires all functional domains of MPP1. A, Scheme of MPP1 functional domains, and of the two MPP1 fragments 1–267 and 268–466, which were tested. B, Cellular fluorescence peak intensities at an emission wavelength of 527 nm were determined of AGTR1eYFP-expressing HEK cells without (-) and with (+) co- transfection of MPP1-encoding plasmid, MPP1-(1–267)-encoding plasmid, MPP1-(268–466)-encoding plasmid, or MPP1-(1–267) and MPP1-(268–466)- encoding plasmids together. Data are presented as mean values ± s.d. (n = 4 biological replicates). P-values were determined by Tukey’s test.

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Functional Assay, Fluorescence, Expressing, Cotransfection, Plasmid Preparation

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 8. Deletion of a putative internal PDZ domain-binding motif in AGTR1-(1–319)-(Δ213-220)-eYFP abolishes the AGTR1-enhancing effect by MPP1 in HEK cells. A, Topological scheme of the AGTR1-(1–359) protein sequence, in which deletions made in construct AGTR1-(1–319)-(Δ213-220) are marked in red. The scheme was drawn with Protter, version 1.0. Residues 213–220 at the beginning of the third intracellular loop of AGTR1 include the sequence “Y-T-L-I”, which could be an internal PDZ domain-binding motif, which is defined by “X-S/T-X-ϕ“ where “X” can be any amino acid, and “ϕ“ is a hydrophobic amino acid. B, Cellular fluorescence peak intensities at an emis sion wavelength of 527 nm were determined of HEK cells without (-) and with stable MPP1 (+) expression, and transfection of AGTR1-(1–319)-eYFP, or AGTR1-(1–319)-(Δ213-220)-eYFP with deletion of a putative internal PDZ domain-binding motif (Δ213-220). Data are presented as mean values ± s.d. (n = 3 biological replicates). P-values were determined by Tukey’s test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Binding Assay, Sequencing, Construct, Fluorescence, Expressing, Transfection

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 10. Upregulation of cardiac Mpp1 transcript levels by diabetes- induced cardiac dysfunction and by Hdac3 deficiency in rodents. A, Car diac Mpp1 transcript levels were up-regulated in rats with diabetes-induced cardiac dysfunction. Data were retrieved from the GEO profile GDS3153 (31), probe set ID 1389963_at of the Affymetrix Rat Expression 230A Array. Hearts were obtained from 12-week-old rats with four weeks of streptozotocin-induced diabetes and from control rats (mean ± s.d., n = 3 hearts per group). B, Upregulation of cardiac Mpp1 in hearts from 6-week-old mice with Hdac3- deficiency (Hdac3 KO) in heart and skeletal muscle (HDAC3fl/fl/MCK-Cre), which develop a severe hypertrophic cardiomyopathy on a high fat diet (32). Control hearts were isolated from wild-type mice (HDAC3fl/fl). Data were taken from the GEO profile GDS4886, probe set ID 106447481 of the Affymetrix Mouse Gene 1.0 ST Array (mean ± s.d., n = 4 male mice per group). P-values were determined by the unpaired, two-tailed t-test.

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Expressing, Control, Isolation, Two Tailed Test

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 11. Detection of increased MPP1 transcript levels in peripheral blood mononuclear cells of old human research participants. A-F, Transcript levels of MPP1 (A), GRK2 (B), GRK3 (C), DUSP3 (D), LRRN3 (E), and CD27 (F) in PBMC from old (age: 75–89 years, y; n = 5) human research participants were determined by whole genome microarray gene expression profiling. PBMC isolated from middle-aged research participants (age: 35–50 years, y; n = 4) served as the control group. Data are shown as mean values ± s.d. P-values were determined by the two- tailed (A,B,D,E,F), or one-tailed (C), unpaired t-test.

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Microarray, Gene Expression, Isolation, Control, Two Tailed Test, One-tailed Test