Journal:

Article Title: Characteristics and Dynamics of Bacterial Populations during Postantibiotic Effect Determined by Flow Cytometry

doi:

Figure Lengend Snippet: MICs, multiples of MICs, and duration of PAEs for organism-antibiotic combinations studied by flow cytometry

Article Snippet: Growth curves from typical PAE experiments with ceftriaxone and ciprofloxacin (each at a concentration equivalent to twice the MIC) against E. coli are shown in Fig. A. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Organism Antibiotic MIC (μg/ml) Multiple of MIC PAE (h) E. coli ATCC 25922 Ampicillin 2.0 2 −0.3 4 −0.4 8 −0.3 Ceftriaxone 0.03 2 −0.3 4 −0.2 8 −0.5 Gentamicin 1.0 1 0.7 2 0.6 Ciprofloxacin 0.015 1 1.5 2 2.7 Rifampin 16 1 0.6 2 1.5 P. aeruginosa ATCC 27853 Imipenem 2.0 2 1.3 4 1.9 8 2.6 Ciprofloxacin 0.5 2 0.7 Open in a separate window MICs, multiples of MICs, and duration of PAEs for organism-antibiotic combinations studied by flow cytometry fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 1 caption a7 (A) A typical PAE experiment for E. coli ATCC 25922 after exposure to ceftriaxone or ciprofloxacin (each at a concentration equivalent to twice the MIC), as determined by viability counting.

Techniques:

Journal:

Article Title: Characteristics and Dynamics of Bacterial Populations during Postantibiotic Effect Determined by Flow Cytometry

doi:

Figure Lengend Snippet: (A) A typical PAE experiment for E. coli ATCC 25922 after exposure to ceftriaxone or ciprofloxacin (each at a concentration equivalent to twice the MIC), as determined by viability counting. As shown, no PAE was seen after ceftriaxone exposure, but ciprofloxacin induced a PAE of 1.9 h. The organisms were stained with propidium iodide and examined by fluorescence microscopy. (B through D) Photomicrographs of untreated control organisms (B), ceftriaxone-exposed organisms 35 min after drug removal (C), and ciprofloxacin-exposed organisms 270 min after drug removal (D). Both antibiotics induced filamentation, but this morphological form persisted past the classically defined PAE in organisms exposed to ciprofloxacin. Magnification, ×880.

Article Snippet: Growth curves from typical PAE experiments with ceftriaxone and ciprofloxacin (each at a concentration equivalent to twice the MIC) against E. coli are shown in Fig. A. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Organism Antibiotic MIC (μg/ml) Multiple of MIC PAE (h) E. coli ATCC 25922 Ampicillin 2.0 2 −0.3 4 −0.4 8 −0.3 Ceftriaxone 0.03 2 −0.3 4 −0.2 8 −0.5 Gentamicin 1.0 1 0.7 2 0.6 Ciprofloxacin 0.015 1 1.5 2 2.7 Rifampin 16 1 0.6 2 1.5 P. aeruginosa ATCC 27853 Imipenem 2.0 2 1.3 4 1.9 8 2.6 Ciprofloxacin 0.5 2 0.7 Open in a separate window MICs, multiples of MICs, and duration of PAEs for organism-antibiotic combinations studied by flow cytometry fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 1 caption a7 (A) A typical PAE experiment for E. coli ATCC 25922 after exposure to ceftriaxone or ciprofloxacin (each at a concentration equivalent to twice the MIC), as determined by viability counting.

Techniques: Concentration Assay, Staining, Fluorescence, Microscopy, Control

Journal:

Article Title: Characteristics and Dynamics of Bacterial Populations during Postantibiotic Effect Determined by Flow Cytometry

doi:

Figure Lengend Snippet: Histograms showing a comparison of the size distribution (FSC-H) (left panels) and nucleic acid content (FL2-H) (middle panels) of E. coli during the PAE after exposure to ampicillin at a concentration equivalent to twice the MIC at 35 min after drug removal and after exposure to rifampin at a concentration equivalent to the MIC (lower panels) at 90 min after drug removal. Dotted-and-dashed lines, control organisms; solid lines, organisms previously exposed to the antibiotics. (Upper right graph) Progressive changes in size, compared to sizes of control organisms, as a function of time after previous exposure to ampicillin. (Lower right graph) Summary of the minimal changes in size that were noted after previous exposure to rifampin. The sizes of the antibiotic-treated organisms were compared to three size intervals derived from the control, which are described in text and shown in Fig. ​Fig.2.2. Open circles, bacteria in the PAE phase which were within 2 SDs of control size; solid squares, bacteria within 2 to 4 SDs; open squares, organisms >4 SDs from the control distribution.

Article Snippet: Growth curves from typical PAE experiments with ceftriaxone and ciprofloxacin (each at a concentration equivalent to twice the MIC) against E. coli are shown in Fig. A. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Organism Antibiotic MIC (μg/ml) Multiple of MIC PAE (h) E. coli ATCC 25922 Ampicillin 2.0 2 −0.3 4 −0.4 8 −0.3 Ceftriaxone 0.03 2 −0.3 4 −0.2 8 −0.5 Gentamicin 1.0 1 0.7 2 0.6 Ciprofloxacin 0.015 1 1.5 2 2.7 Rifampin 16 1 0.6 2 1.5 P. aeruginosa ATCC 27853 Imipenem 2.0 2 1.3 4 1.9 8 2.6 Ciprofloxacin 0.5 2 0.7 Open in a separate window MICs, multiples of MICs, and duration of PAEs for organism-antibiotic combinations studied by flow cytometry fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 1 caption a7 (A) A typical PAE experiment for E. coli ATCC 25922 after exposure to ceftriaxone or ciprofloxacin (each at a concentration equivalent to twice the MIC), as determined by viability counting.

Techniques: Comparison, Concentration Assay, Control, Derivative Assay, Bacteria

Journal:

Article Title: Characteristics and Dynamics of Bacterial Populations during Postantibiotic Effect Determined by Flow Cytometry

doi:

Figure Lengend Snippet: Histograms showing a comparison of the size distributions (FSC-H; left panels) and nucleic acid contents (FL2-H; middle panels) of E. coli during the PAE after exposure to ciprofloxacin at a concentration equivalent to the MIC and at a concentration equivalent to twice the MIC at 270 min after drug removal. Dotted-and-dashed lines, control organisms; solid lines, bacteria previously exposed to ciprofloxacin. Graphs (right panels) show progressive changes in size compared to sizes of controls. The sizes of antibiotic-treated organisms were compared to three control size intervals described in the text and shown in Fig. ​Fig.2.2. Open circles, bacteria within 2 SDs of control size; solid squares, bacteria within 2 to 4 SDs; open squares, organisms >4 SDs from the control distribution.

Article Snippet: Growth curves from typical PAE experiments with ceftriaxone and ciprofloxacin (each at a concentration equivalent to twice the MIC) against E. coli are shown in Fig. A. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Organism Antibiotic MIC (μg/ml) Multiple of MIC PAE (h) E. coli ATCC 25922 Ampicillin 2.0 2 −0.3 4 −0.4 8 −0.3 Ceftriaxone 0.03 2 −0.3 4 −0.2 8 −0.5 Gentamicin 1.0 1 0.7 2 0.6 Ciprofloxacin 0.015 1 1.5 2 2.7 Rifampin 16 1 0.6 2 1.5 P. aeruginosa ATCC 27853 Imipenem 2.0 2 1.3 4 1.9 8 2.6 Ciprofloxacin 0.5 2 0.7 Open in a separate window MICs, multiples of MICs, and duration of PAEs for organism-antibiotic combinations studied by flow cytometry fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 1 caption a7 (A) A typical PAE experiment for E. coli ATCC 25922 after exposure to ceftriaxone or ciprofloxacin (each at a concentration equivalent to twice the MIC), as determined by viability counting.

Techniques: Comparison, Concentration Assay, Control, Bacteria

Journal:

Article Title: Characteristics and Dynamics of Bacterial Populations during Postantibiotic Effect Determined by Flow Cytometry

doi:

Figure Lengend Snippet: Histograms showing the size distributions (FSC-H; left panels) and nucleic acid contents (FL2-H; middle panels) of P. aeruginosa during the PAE after exposure to imipenem at a concentration equivalent to twice the MIC and to ciprofloxacin at a concentration equivalent to the MIC at 180 and 70 min after drug removal, respectively. Dotted-and-dashed lines, control organisms; solid lines, antibiotic-exposed organisms. The graphs (right panels) show progressive changes in size compared to sizes of controls. The sizes of antibiotic-treated organisms were compared to three control size intervals described in text and shown in Fig. ​Fig.2.2. Open circles, bacteria within 2 SDs of control size; solid squares, bacteria within 2 to 4 SDs; open squares, organisms >4 SDs from the control distribution.

Article Snippet: Growth curves from typical PAE experiments with ceftriaxone and ciprofloxacin (each at a concentration equivalent to twice the MIC) against E. coli are shown in Fig. A. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Organism Antibiotic MIC (μg/ml) Multiple of MIC PAE (h) E. coli ATCC 25922 Ampicillin 2.0 2 −0.3 4 −0.4 8 −0.3 Ceftriaxone 0.03 2 −0.3 4 −0.2 8 −0.5 Gentamicin 1.0 1 0.7 2 0.6 Ciprofloxacin 0.015 1 1.5 2 2.7 Rifampin 16 1 0.6 2 1.5 P. aeruginosa ATCC 27853 Imipenem 2.0 2 1.3 4 1.9 8 2.6 Ciprofloxacin 0.5 2 0.7 Open in a separate window MICs, multiples of MICs, and duration of PAEs for organism-antibiotic combinations studied by flow cytometry fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 1 caption a7 (A) A typical PAE experiment for E. coli ATCC 25922 after exposure to ceftriaxone or ciprofloxacin (each at a concentration equivalent to twice the MIC), as determined by viability counting.

Techniques: Concentration Assay, Control, Bacteria

Journal: Leukemia

Article Title: P4HA2 hydroxylates SUFU to regulate the paracrine Hedgehog signaling and promote B-cell lymphoma progression.

doi: 10.1038/s41375-024-02313-8

Figure Lengend Snippet: Fig. 1 P4HA2 interacts with KIF7 and regulates the Hedgehog signaling. A P4HA2 interacts with KIF7. HEK293T cells were transfected with Flag-tagged P4HA2. Cell lysates were prepared and subjected to immunoprecipitation (IP) with M2 anti-Flag beads. The immunoprecipitates were analyzed by Western blotting (WB) with anti-Flag and KIF7 antibodies. B KIF7 interacts with P4HA2. HEK293T cells were transfected with Flag-tagged KIF7. Cell lysates were prepared and subjected to Flag IP. The immunoprecipitants were analyzed by WB with anti-Flag and P4HA2 antibodies. C Schematic representation of the SHH signaling pathway. SAG is a chlorobenzothiophene-containing Hh pathway agonist and binds to the SMO heptahelical bundle in a manner that antagonizes cyclopamine action. SAG activates SMO, leading to the release of GLI1 by SUFU, and the Hh signaling pathway is then activated. D–I Knockout of P4HA2 causes suppression of Hedgehog signal transduction. NIH/3T3 (D–F) and OP9 (G–I) P4HA2 knockout (KO) or vehicle cells were treated with (+) or without (−) 200 nM SAG. The Hh targeted genes, Gli1 (E, H) and Ptch1 (F, I), were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Data are shown as the mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. All experiments were repeated three times independently.

Article Snippet: The following antibodies were used for western blotting (WB), immunofluorescence (IF): PAN-OH (home-made; 1:1,000 for WB); P4HA2 (13759-1- AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC, 1:200 for IF); P4HA1 (12658-1-AP, rabbit, Proteintech; 1:1,000 for WB); KIF7 (24693-1-AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC); SUFU (26759-1-AP, rabbit, Proteintech; 1:2,000 for WB); Flag (clone M2, F1804, mouse, Sigma-Aldrich; 1:10,000 for WB, 1:200 for IF); His (clone OGHis, D291-3, mouse, Medical & Biological Laboratories; 1:5,000 for WB); V5 (AB3792, rabbit, Merck Millipore; 1:2,000 for WB); GLI1 (clone A-7, sc-515781, mouse, Santa Cruz; 1:1,000 for WB, 1:200 for IHC); GLI2 (18989-1-AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC) ; GAPDH (clone 1E6D9, 60004-1-Ig, mouse, Proteintech; 1:10,000 for WB); Arl13b (clone N295B/66, 75-287, mouse, NeuroMab; 1:200 for IF); Cy3 AffiniPure Goat Anti-Mouse IgG (H+ L) (115- 165-003, Jackson ImmunoResearch; 1:10,000 for IF); α-SMA (#34105, Cell Signaling Technology, 1:100 for IF) Light chain specific Mouse Anti-Rabbit IgG (211-032-171, Jackson ImmunoResearch; 1:10,000 for WB); Light chain specific Goat Anti Mouse IgG (115-035-174, Jackson ImmunoResearch; 1:10,000 for WB); Peroxidase AffiniPure Goat Anti-Rabbit IgG, Fc fragment specific (111-035-008, Jackson ImmunoResearch; 1:10,000 for WB); Donkey anti-mouse IgG (H&L) (715-035-150, Jackson ImmunoResearch; 1:10,000 for WB); Goat Anti-Rabbit IgG (H+ L) (111-035-003, Jackson ImmunoResearch; 1:10,000 for WB); Peroxidase AffiniPure Goat Anti-Mouse IgG, Fcγ fragment specific (115-035-008, Jackson ImmunoResearch; 1:10,000 for WB); Smoothened Agonist (SAG), Selleck, Cat# S7779; Fetal bovine serum, Thermo Fisher, Cat# 16140071; Penicillin/streptomycin, Thermo Fisher, Cat# 15140122; RPMI 1640, Thermo Fisher, Cat# 11875093; DMEM, Thermo Fisher, Cat# 11995065; Puromycin Dihydrochloride, Sigma, Cat# P8833; Trypsin 0.25% EDTA, Thermo Fisher, Cat# 25200072; Polyethylenimine Hydrochloride (MW 40,000), Polysciences, Cat# 24765-1; Polybrene Transfection Reagent, Sigma, Cat# TR-1003-G; SHH protein, Yun Zhao Laboratory; Anti-V5-tag mAb-Magnetic Beads, Medical & Biological Laboratories, Cat# M215-11; NTI-FLAG® M2 Affinity Gel, Sigma, Cat# F2426; Ni-NTA Agarose, QIAGEN, Cat# 30230; HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper), Vazyme, Cat# R212-01/02; ChamQ SYBR qPCR Master Mix, Vazyme, Cat# Q311-02.

Techniques: Transfection, Immunoprecipitation, Western Blot, Knock-Out, Transduction, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Leukemia

Article Title: P4HA2 hydroxylates SUFU to regulate the paracrine Hedgehog signaling and promote B-cell lymphoma progression.

doi: 10.1038/s41375-024-02313-8

Figure Lengend Snippet: Fig. 3 P4HA2 migrates with KIF7 to regulate the Hedgehog pathway transduction. A The trafficking of the P4HA2-KIF7 complex from the cytoplasm to the cilium responds to Hedgehog signaling. NIH/3T3 cells were co-infected with KIF7-BFP and P4HA2-EGFP lentivirus. White arrow indicates the co-localization of cilia, KIF7-BFP and P4HA2-EGFP. Yellow arrow indicates the co-localization of cilia and P4HA2-EGFP, which may bind with endogenous KIF7. Cells were treated with 200 nM SAG (+) or not (−). Cells were fixed and stained with antibodies for ARL13B to mark primary cilia. Scale bars: 5 μm. B Statistical analysis of the relative colocalization rate in (A) indicated cells. Data are shown as the mean ± SEM; SAG (−), n = 11; SAG (+), n = 13. **P < 0.01. C The regulation of the Hedgehog signaling by P4ha2 is dependent on KIF7. KIF7 knockout (KO) and the control cells were knocked down with P4HA2 (shP4HA2). Cells were treated with (+) or without (−) 200 nM SAG. qRT- PCR analysis of GLI1 transcripts was performed using RNA isolated from the indicated cell lines. Data are shown as the mean ± SEM (n = 3). ***P < 0.001. All experiments were repeated three times independently.

Article Snippet: The following antibodies were used for western blotting (WB), immunofluorescence (IF): PAN-OH (home-made; 1:1,000 for WB); P4HA2 (13759-1- AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC, 1:200 for IF); P4HA1 (12658-1-AP, rabbit, Proteintech; 1:1,000 for WB); KIF7 (24693-1-AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC); SUFU (26759-1-AP, rabbit, Proteintech; 1:2,000 for WB); Flag (clone M2, F1804, mouse, Sigma-Aldrich; 1:10,000 for WB, 1:200 for IF); His (clone OGHis, D291-3, mouse, Medical & Biological Laboratories; 1:5,000 for WB); V5 (AB3792, rabbit, Merck Millipore; 1:2,000 for WB); GLI1 (clone A-7, sc-515781, mouse, Santa Cruz; 1:1,000 for WB, 1:200 for IHC); GLI2 (18989-1-AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC) ; GAPDH (clone 1E6D9, 60004-1-Ig, mouse, Proteintech; 1:10,000 for WB); Arl13b (clone N295B/66, 75-287, mouse, NeuroMab; 1:200 for IF); Cy3 AffiniPure Goat Anti-Mouse IgG (H+ L) (115- 165-003, Jackson ImmunoResearch; 1:10,000 for IF); α-SMA (#34105, Cell Signaling Technology, 1:100 for IF) Light chain specific Mouse Anti-Rabbit IgG (211-032-171, Jackson ImmunoResearch; 1:10,000 for WB); Light chain specific Goat Anti Mouse IgG (115-035-174, Jackson ImmunoResearch; 1:10,000 for WB); Peroxidase AffiniPure Goat Anti-Rabbit IgG, Fc fragment specific (111-035-008, Jackson ImmunoResearch; 1:10,000 for WB); Donkey anti-mouse IgG (H&L) (715-035-150, Jackson ImmunoResearch; 1:10,000 for WB); Goat Anti-Rabbit IgG (H+ L) (111-035-003, Jackson ImmunoResearch; 1:10,000 for WB); Peroxidase AffiniPure Goat Anti-Mouse IgG, Fcγ fragment specific (115-035-008, Jackson ImmunoResearch; 1:10,000 for WB); Smoothened Agonist (SAG), Selleck, Cat# S7779; Fetal bovine serum, Thermo Fisher, Cat# 16140071; Penicillin/streptomycin, Thermo Fisher, Cat# 15140122; RPMI 1640, Thermo Fisher, Cat# 11875093; DMEM, Thermo Fisher, Cat# 11995065; Puromycin Dihydrochloride, Sigma, Cat# P8833; Trypsin 0.25% EDTA, Thermo Fisher, Cat# 25200072; Polyethylenimine Hydrochloride (MW 40,000), Polysciences, Cat# 24765-1; Polybrene Transfection Reagent, Sigma, Cat# TR-1003-G; SHH protein, Yun Zhao Laboratory; Anti-V5-tag mAb-Magnetic Beads, Medical & Biological Laboratories, Cat# M215-11; NTI-FLAG® M2 Affinity Gel, Sigma, Cat# F2426; Ni-NTA Agarose, QIAGEN, Cat# 30230; HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper), Vazyme, Cat# R212-01/02; ChamQ SYBR qPCR Master Mix, Vazyme, Cat# Q311-02.

Techniques: Transduction, Infection, Staining, Knock-Out, Control, Quantitative RT-PCR, Isolation

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

Journal: eLife

Article Title: A zebrafish embryo screen utilizing gastrulation identifies the HTR2C inhibitor pizotifen as a suppressor of EMT-mediated metastasis

doi: 10.7554/eLife.70151

Figure Lengend Snippet: The analyses were performed by ImageJ. Signal strength of bands of E-cadherin, EpCAM, KRT18, KRT19, MMP1, MMP3, vimentin, and S100A4 was normalized by that of GAPDH.

Article Snippet: Anti-phospho-GSK3β (Ser9) (F-2), anti-GSK3β (1F7), anti-KRT18 (DC-10), anti-KRT19 (A53-B/A2), anti-MMP1 (3B6), anti-MMP2 (8B4), anti-S100A4 (A-7), anti-luciferase (C-12), anti-ki67 (ki-67), and anti-β-catenin (E-5) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques:

Journal: eLife

Article Title: A zebrafish embryo screen utilizing gastrulation identifies the HTR2C inhibitor pizotifen as a suppressor of EMT-mediated metastasis

doi: 10.7554/eLife.70151

Figure Lengend Snippet:

Article Snippet: Anti-phospho-GSK3β (Ser9) (F-2), anti-GSK3β (1F7), anti-KRT18 (DC-10), anti-KRT19 (A53-B/A2), anti-MMP1 (3B6), anti-MMP2 (8B4), anti-S100A4 (A-7), anti-luciferase (C-12), anti-ki67 (ki-67), and anti-β-catenin (E-5) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Luciferase, Recombinant, Expressing, Plasmid Preparation, Software

Journal: International Journal of Molecular Sciences

Article Title: Ischemic Rescue Potential of Conditioned Medium Derived from Skeletal Muscle Cells-Seeded Electrospun Fiber-Coated Human Amniotic Membrane Scaffolds

doi: 10.3390/ijms252111697

Figure Lengend Snippet: The effects of CM on HCM subjected to H/R-induced injury according to the XIAP expression profile. Immunofluorescence images of HCM stained with XIAP antibody (red) and DAPI (blue) after being treated with different CM following H/R-induced injury (10× magnification; 200 µm scale bar; n = 3). The bar graph represents the MFI measurement for XIAP in H/R-injured HCM after ischemic rescue with CM. Treatment with different EF–HAM CM did not upregulate the expression of XIAP injured HCM. The values are represented as mean ± SEM ( n = 3; * p < 0.05 or ** p < 0.01, compared with Normoxic control; one-way ANOVA).

Article Snippet: The HCMs were incubated with mouse monoclonal anti-human XIAP (A-7) antibody (1:50 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), followed by immunolabeling by goat anti-mouse Alexa Fluor ® 594 secondary antibody (1:300 dilution) and counterstaining with DAPI.

Techniques: Expressing, Immunofluorescence, Staining, Control