human mpp1  (Santa Cruz Biotechnology)

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: EJHaem

Article Title: A novel cause of DKC1 ‐related bone marrow failure: Partial deletion of the 3′ untranslated region

doi: 10.1002/jha2.165

Figure Lengend Snippet: Deletion in the DKC1 3′ UTR segregates with disease phenotype. A, Location of the 1104 bp deletion on chromosome X, encompassing part of the DKC1 3′ UTR and the intergenic region between genes DKC1 and MPP1 . B, Magnification of the deleted region, showing the location of polyadenylation signals (PolyA; confirmed in Figure ) and primers used for PCR and sequencing (A‐fwd, B‐rev, C‐seq). C, Sanger sequencing (using primer C‐seq) of the PCR product from primers A‐fwd and B‐rev, in the proband (V‐2) and his father (IV‐2). Black arrow marks the 5′ end of the deletion. D, Agarose gel of products of PCR across the deletion in 12 family members. The proband is indicated in blue; shown in red are female family members with signs of disease. The sizes of the wild‐type (WT) or deleted (ΔUTR) PCR products are shown on the right. Note that DKC1 is on the X chromosome, so male individuals (III‐2, IV‐2, V‐2) carry a single allele

Article Snippet: A portion (2.5%) of the cDNA was used for real‐time PCR with Platinum SYBR Green qPCR SuperMix‐UDG (Invitrogen) and 400 nM primers DKFA and DKRA for DKC1 , hTR‐F and hTR‐R for TERC , or a qSTAR qPCR primer pair for MPP1 (OriGene Technologies; Table S1).

Techniques: Sequencing, Agarose Gel Electrophoresis

Journal: EJHaem

Article Title: A novel cause of DKC1 ‐related bone marrow failure: Partial deletion of the 3′ untranslated region

doi: 10.1002/jha2.165

Figure Lengend Snippet: Short telomeres correlate with presence of the DKC1 deletion. Telomere lengths of 12 family members, measured by qPCR (A) or TRF Southern blot analysis (B). The proband is indicated in blue; shown in red are heterozygous female family members. Curves in (A) represent the indicated percentiles of telomere lengths in ∼240 healthy individuals. Mean TRF lengths in kb are indicated below each lane of the Southern blot in (B)

Article Snippet: A portion (2.5%) of the cDNA was used for real‐time PCR with Platinum SYBR Green qPCR SuperMix‐UDG (Invitrogen) and 400 nM primers DKFA and DKRA for DKC1 , hTR‐F and hTR‐R for TERC , or a qSTAR qPCR primer pair for MPP1 (OriGene Technologies; Table S1).

Techniques: Southern Blot

Journal: EJHaem

Article Title: A novel cause of DKC1 ‐related bone marrow failure: Partial deletion of the 3′ untranslated region

doi: 10.1002/jha2.165

Figure Lengend Snippet: The proband expresses very low levels of DKC1 mRNA and telomerase RNA. A, RT‐PCR of DKC1 transcripts in peripheral blood cells of the proband (V‐2) and his immediate family members, and RNA from HeLa cells or an unrelated individual (Ctrl) as wild‐type controls. −RT: negative control in the absence of reverse transcriptase. B, A second round of nested PCR was performed on the products from (A), using an internal primer. C, Levels of DKC1 mRNA, measured by quantitative real‐time RT‐PCR (RT‐qPCR) using random hexamers for cDNA transcription in the proband (V‐2) and his immediate family members. Female relatives heterozygous for the deletion are labeled in red. Data shown as mean ± SEM of expression levels relative to individual IV‐2; **** P < .0001, as determined by two‐way ANOVA followed by Tukey's multiple comparison tests; n = 3‐4 independent RT reactions from each of two blood samples. D, Levels of the telomerase RNA subunit, hTR, measured by RT‐qPCR using random hexamers for cDNA transcription in the proband (V‐2) and his immediate family members. Female relatives heterozygous for the deletion are labeled in red. Data shown as mean ± SEM of expression levels relative to individual IV‐2; **** P < .0001, as determined by two‐way ANOVA followed by Tukey's multiple comparison tests; n = 5 independent RT reactions from each of two blood samples

Article Snippet: A portion (2.5%) of the cDNA was used for real‐time PCR with Platinum SYBR Green qPCR SuperMix‐UDG (Invitrogen) and 400 nM primers DKFA and DKRA for DKC1 , hTR‐F and hTR‐R for TERC , or a qSTAR qPCR primer pair for MPP1 (OriGene Technologies; Table S1).

Techniques: Reverse Transcription Polymerase Chain Reaction, Negative Control, Reverse Transcription, Nested PCR, Quantitative RT-PCR, Labeling, Expressing, Comparison

Journal: Molecular Biology of the Cell

Article Title: Kinesin-6 KIF20B is required for efficient cytokinetic furrowing and timely abscission in human cells

doi: 10.1091/mbc.E17-08-0495

Figure Lengend Snippet: KIF20B localizes to the central spindle and midbody throughout cytokinesis in HeLa cells. Panels A–I show immunofluorescence staining for endogenous KIF20B (green) and tubulin (red), at sequential phases of cytokinesis from anaphase furrowing (A) to postabscission (I). Arrowheads point to the central dark zone in all pictures. (A–B″) During anaphase, KIF20B starts to accumulate as speckles along the microtubules of the central spindle (A′), and in later furrows forming a dense band in the middle of the central spindle (B′). (C–D″) In early midbodies, KIF20B accumulates on the inner flanks of the midbody surrounding the dark zone (arrowhead), forming a cap-like structure. (E–F″) In late midbody stage, KIF20B spreads out on the midbody flanks surrounding the constriction sites, resulting in four distinct spots of KIF20B localization. (G–I″) Near abscission in very thin midbodies, small spots of KIF20B can be seen still surrounding the central dark zone (arrowhead). The small arrows in the G panels point to KIF20B dots localizing along a strand of microtubules. (J–J″) GFP-KIF20B expressed in HeLa cell shows the same localization within the midbody as detected by antibodies to KIF20B. Scale bars represent 5 µm for A–I″ and 10 µm for panel J.

Article Snippet: Primary antibodies used were as follows: mouse monoclonal DM1α (α-tubulin; 1:500) was from Abcam; rat anti-TUBA1A (clone YL ½; 1:750) was from Novus Biologicals; mouse polyclonal anti-CEP55 (1:200) was from Abnova; mouse anti-Aurora kinase B (AURKB; 1:300) was from BD Biosciences; rabbit anti-phospho-T232-Aurora kinase B (pAURKB; 1:200) was from Rockland; rabbit anti-KIF20A (A300-879A; 1:100) was from Bethyl Labs; goat anti-anillin (ANLN; 1:300), mouse monoclonal anti-ANLN (1:100), rabbit anti-MKLP1 (sc-867; 1:100), rabbit anti-PRC1 (1:50), mouse monoclonal anti-human-spastin (3G11/1; 1:50), and mouse anti-human-MPP1(KIF20B; 1:300) were from Santa Cruz; rabbit anti-mouse-Kif20b (1:500) was custom-made by Covance ( Janisch, Vock, et al. , 2013 ); rabbit anti-cleaved-caspase 3 (CC3; 1:200) and rabbit anti-phosphohistone H3 (PH3, Alexa Fluor 647 conjugated; 1:400) were from Cell Signaling; rabbit anti-α-ACTININ4 (ACTN4; 1:250) was from Millipore; and rabbit anti-VPS4 (1:500) was from Sigma Aldrich.

Techniques: Immunofluorescence, Staining

Journal: Molecular Biology of the Cell

Article Title: Kinesin-6 KIF20B is required for efficient cytokinetic furrowing and timely abscission in human cells

doi: 10.1091/mbc.E17-08-0495

Figure Lengend Snippet: KIF20B protein shows overlapping but distinct localization from MKLP1/KIF23 and MKLP2/KIF20A during cytokinesis. (A) Immunostaining HeLa cells for endogenous MKLP1 (red) and KIF20B (green) with α-tubulin (white) shows distinct localizations at prefurrow (a), early furrow (b), early midbody (c), and late midbody (d) stages. (B) Immunostaining for endogenous MKLP2 (red) and KIF20B (green) with α-tubulin (white) shows substantial overlap in the early furrow (b) and early midbody (c) stages, but that KIF20B is more broadly distributed on the central spindle in the prefurrow stage (a) and is enriched on the outer flanks of the constriction sites in the late midbody stage (d).

Article Snippet: Primary antibodies used were as follows: mouse monoclonal DM1α (α-tubulin; 1:500) was from Abcam; rat anti-TUBA1A (clone YL ½; 1:750) was from Novus Biologicals; mouse polyclonal anti-CEP55 (1:200) was from Abnova; mouse anti-Aurora kinase B (AURKB; 1:300) was from BD Biosciences; rabbit anti-phospho-T232-Aurora kinase B (pAURKB; 1:200) was from Rockland; rabbit anti-KIF20A (A300-879A; 1:100) was from Bethyl Labs; goat anti-anillin (ANLN; 1:300), mouse monoclonal anti-ANLN (1:100), rabbit anti-MKLP1 (sc-867; 1:100), rabbit anti-PRC1 (1:50), mouse monoclonal anti-human-spastin (3G11/1; 1:50), and mouse anti-human-MPP1(KIF20B; 1:300) were from Santa Cruz; rabbit anti-mouse-Kif20b (1:500) was custom-made by Covance ( Janisch, Vock, et al. , 2013 ); rabbit anti-cleaved-caspase 3 (CC3; 1:200) and rabbit anti-phosphohistone H3 (PH3, Alexa Fluor 647 conjugated; 1:400) were from Cell Signaling; rabbit anti-α-ACTININ4 (ACTN4; 1:250) was from Millipore; and rabbit anti-VPS4 (1:500) was from Sigma Aldrich.

Techniques: Immunostaining

Journal: Molecular Biology of the Cell

Article Title: Kinesin-6 KIF20B is required for efficient cytokinetic furrowing and timely abscission in human cells

doi: 10.1091/mbc.E17-08-0495

Figure Lengend Snippet: Cytokinesis defects in KIF20B-depleted asynchronous HeLa cell cultures. (A) The average mitotic index was not altered by KIF20B depletion at 24 h ( n = 5 coverslips/treatment, siLUC = 1192 cells, siKIF = 1408 cells) or 48 h posttransfection ( n = 7 coverslips/treatment, siLUC = 1870 cells, siKIF = 2513 cells). Mitotic index was defined as the number of phosphohistone H3 positive (PH3 + ) cells divided by the total cell count by DAPI+ nuclei. PH3 immunostaining signal is strong in prophase, metaphase, and anaphase; weak in telophase; and absent in posttelophase late midbody stage cells. (B) Average percentage of telophase cells out of mitotic cells (PH3+) was not changed at 24 h but decreased at 48 h posttransfection in siKIF knockdown cells ( p = 0.038). Telophase was characterized by the presence of condensed chromatin. For 24 h, n = 5 coverslips/treatment (siLUC = 67 cells; siKIF = 95 cells); for 48 h posttransfection, n = 7 coverslips/treatment (siLUC = 49 cells; siKIF = 69 cells). (C) The average percentage of midbody stage cells out of all dividing cells (PH3+ or PH3− with a midbody) was not significantly different in siKIF-treated cells. At 24 h, n = 8 coverslips/treatment, with 2556 total siLUC cells, and 3048 total siKIF cells; at 48 h posttransfection, n = 6 coverslips/treatment, with 952 total siLUC cells, and 1176 total siKIF cells). (D) The percentage of multinucleate cells out of total cells was significantly increased in the KIF20B-depleted cultures at 24 h posttransfection ( p = 0.005). For 24 h, n = 5 coverslips/treatment with 1192 total siLUC cells and 1408 total siKIF cells. For 48 h, n = 6 coverslips/treatment, with 1870 total siLUC cells and 2513 total siKIF cells). (E, E′) A control siLUC HeLa cell stained with α-tubulin (TUBA1A) and DAPI with a single nucleus. (F, F′) siKIF transfected cell stained with TUBA1A and DAPI showing two nuclei within the same cell (white arrows). (G) The percentage of cells with multi-lobed nuclei was significantly greater in the knockdown cells than in the control cells ( p = 4.77 × 10 −5 ) at 48 h posttransfection. For 24 h, n = 5 coverslips/treatment, with 1192 total siLUC cells and 1408 total siKIF cells. For 48 h, n = 6 coverslips/treatment, with 1870 total siLUC cells and 2513 siKIF cells. (H, H′) siKIF transfected cells stained with TUBA1A and DAPI to show multi-lobed phenotype. White arrows point at constrictions within single nuclei. (I) Average apoptotic index (cleaved-caspase-3 CC3+ out of total cells) was increased after siKIF treatment ( p = 0.006). n = 8 coverslips/treatment, with 1878 siLUC cells and 1486 siKIF cells. Coverslips were prepared from three independent siRNA transfection experiments. p Values stated for Student’s t test.

Article Snippet: Primary antibodies used were as follows: mouse monoclonal DM1α (α-tubulin; 1:500) was from Abcam; rat anti-TUBA1A (clone YL ½; 1:750) was from Novus Biologicals; mouse polyclonal anti-CEP55 (1:200) was from Abnova; mouse anti-Aurora kinase B (AURKB; 1:300) was from BD Biosciences; rabbit anti-phospho-T232-Aurora kinase B (pAURKB; 1:200) was from Rockland; rabbit anti-KIF20A (A300-879A; 1:100) was from Bethyl Labs; goat anti-anillin (ANLN; 1:300), mouse monoclonal anti-ANLN (1:100), rabbit anti-MKLP1 (sc-867; 1:100), rabbit anti-PRC1 (1:50), mouse monoclonal anti-human-spastin (3G11/1; 1:50), and mouse anti-human-MPP1(KIF20B; 1:300) were from Santa Cruz; rabbit anti-mouse-Kif20b (1:500) was custom-made by Covance ( Janisch, Vock, et al. , 2013 ); rabbit anti-cleaved-caspase 3 (CC3; 1:200) and rabbit anti-phosphohistone H3 (PH3, Alexa Fluor 647 conjugated; 1:400) were from Cell Signaling; rabbit anti-α-ACTININ4 (ACTN4; 1:250) was from Millipore; and rabbit anti-VPS4 (1:500) was from Sigma Aldrich.

Techniques: Cell Counting, Immunostaining, Staining, Transfection

Journal: Molecular Biology of the Cell

Article Title: Kinesin-6 KIF20B is required for efficient cytokinetic furrowing and timely abscission in human cells

doi: 10.1091/mbc.E17-08-0495

Figure Lengend Snippet: Cleavage furrow ingression is slower in KIF20B-depleted cells. (A, B) Example images of central spindles of furrowing cells fixed with either PFA-MeOH (A) or TCA-CBS (B) and stained for α-tubulin (TUBA1A). Central spindles of some KIF20B-depleted cells appear to have irregularities in the angles or left-right symmetry of the microtubule bundles (arrowheads). (C, D) Representative brightfield time-lapse live images of an siLUC-treated cell (C) that completed furrowing in 6 min and an siKIF-treated cell (D) that completed furrowing in 8 min. (E) Average total time from anaphase onset (chromosome segregation onset) to completion of cleavage furrowing ingression was increased in siKIF cells (****: p = 3 × 10 −7 ). (F) Furrow widths of siKIF cells decreased steadily but more slowly than those of siLUC cells (for points under bracket; ****: p = 3 × 10 −5 ). (G) Cell lengths (pole to pole) of siKIF20B-treated cells also increased steadily but more slowly than in siLUC cells (*: p ≤ 0.05). For E–G, n siLUC = 20 cells and n siKIF = 24 cells across five independent imaging sessions. t = 0 was the last time point before anaphase onset (chromosome segregation) was detectable. Scale bars represent 10 µm.

Article Snippet: Primary antibodies used were as follows: mouse monoclonal DM1α (α-tubulin; 1:500) was from Abcam; rat anti-TUBA1A (clone YL ½; 1:750) was from Novus Biologicals; mouse polyclonal anti-CEP55 (1:200) was from Abnova; mouse anti-Aurora kinase B (AURKB; 1:300) was from BD Biosciences; rabbit anti-phospho-T232-Aurora kinase B (pAURKB; 1:200) was from Rockland; rabbit anti-KIF20A (A300-879A; 1:100) was from Bethyl Labs; goat anti-anillin (ANLN; 1:300), mouse monoclonal anti-ANLN (1:100), rabbit anti-MKLP1 (sc-867; 1:100), rabbit anti-PRC1 (1:50), mouse monoclonal anti-human-spastin (3G11/1; 1:50), and mouse anti-human-MPP1(KIF20B; 1:300) were from Santa Cruz; rabbit anti-mouse-Kif20b (1:500) was custom-made by Covance ( Janisch, Vock, et al. , 2013 ); rabbit anti-cleaved-caspase 3 (CC3; 1:200) and rabbit anti-phosphohistone H3 (PH3, Alexa Fluor 647 conjugated; 1:400) were from Cell Signaling; rabbit anti-α-ACTININ4 (ACTN4; 1:250) was from Millipore; and rabbit anti-VPS4 (1:500) was from Sigma Aldrich.

Techniques: Staining, Imaging

Journal: Molecular Biology of the Cell

Article Title: Kinesin-6 KIF20B is required for efficient cytokinetic furrowing and timely abscission in human cells

doi: 10.1091/mbc.E17-08-0495

Figure Lengend Snippet: Midbodies tend to be thinner in KIF20B-depleted HeLa cells. 24 h after transfection with control (siLUC) or KIF20B (siKIF) siRNA, cells were fixed and immunostained for α-tubulin (TUBA1A) and Aurora B kinase (AURKB). Midbodies were imaged and measured for width and length as shown in A and insets. (A) Schematic of midbody (not to scale) and measurements. The midbody core “dark zone” contains dense overlapping microtubules, but does not stain with tubulin or Aurora B antibodies due to density. Midbody widths were measured adjacent to the dark zone by both tubulin and AURKB signal. Lengths were measured by AURKB signal only. The midbody “edge” was considered to be where no signal was detected. (B, C) Plots of the distributions of midbody widths for KIF20B-depleted cells (siKIF, white bars) and controls (siLUC, black bars) show that KIF20B-depleted cells more often have thin midbodies when measured by TUBA1A or AURKB signal (medians: siLUC = 0.8 µm, siKIF = 0.7 µm, p M-W < 0.0001, and distribution shape p K-S = 0.0205; and siLUC = 1.0 µm, siKIF = 0.8 µm, p M-W < 0.01, and distribution shape p K-S = 0.000419, respectively). (D) Median midbody length, as measured with AURKB signal, shows a trend to be increased in siKIF-treated cells, but does not reach statistical significance ( p M-W = 0.28; p K-S = 0.85). p Values ( p M-W ) for medians are calculated with Mann-Whitney U test; p values ( p K-S ) for distribution shape are calculated with a Kolmogorov-Smirnov test; n.s., not significant. n siLUC = 80 midbodies and n siKIF = 100 midbodies, from three independent siRNA transfections.

Article Snippet: Primary antibodies used were as follows: mouse monoclonal DM1α (α-tubulin; 1:500) was from Abcam; rat anti-TUBA1A (clone YL ½; 1:750) was from Novus Biologicals; mouse polyclonal anti-CEP55 (1:200) was from Abnova; mouse anti-Aurora kinase B (AURKB; 1:300) was from BD Biosciences; rabbit anti-phospho-T232-Aurora kinase B (pAURKB; 1:200) was from Rockland; rabbit anti-KIF20A (A300-879A; 1:100) was from Bethyl Labs; goat anti-anillin (ANLN; 1:300), mouse monoclonal anti-ANLN (1:100), rabbit anti-MKLP1 (sc-867; 1:100), rabbit anti-PRC1 (1:50), mouse monoclonal anti-human-spastin (3G11/1; 1:50), and mouse anti-human-MPP1(KIF20B; 1:300) were from Santa Cruz; rabbit anti-mouse-Kif20b (1:500) was custom-made by Covance ( Janisch, Vock, et al. , 2013 ); rabbit anti-cleaved-caspase 3 (CC3; 1:200) and rabbit anti-phosphohistone H3 (PH3, Alexa Fluor 647 conjugated; 1:400) were from Cell Signaling; rabbit anti-α-ACTININ4 (ACTN4; 1:250) was from Millipore; and rabbit anti-VPS4 (1:500) was from Sigma Aldrich.

Techniques: Transfection, Staining, MANN-WHITNEY

Journal: Molecular Biology of the Cell

Article Title: Kinesin-6 KIF20B is required for efficient cytokinetic furrowing and timely abscission in human cells

doi: 10.1091/mbc.E17-08-0495

Figure Lengend Snippet: Midbody assembly appears normal in KIF20B-depleted cells. (A, B) PRC1 localizes to the central spindle in anaphase in both siLUC and siKIF cells. In early and late midbodies (MB), PRC1 lines the microtubules of the midbody flanks and extends into the cell on the microtubule network. It also forms two distinct disks around the core of the midbody (white arrow). (C, D) MKLP1/KIF23 localizes to the central spindle in anaphase in siLUC and siKIF cells. In early and late midbodies, it is found in the center of the midbody (dark zone) in control or KIF20B depleted cells. By contrast, Aurora kinase B (AURKB) localizes to the flanks of the midbody in early and late stages in both siLUC and siKIF cells. (E, F) Phospho-T232-Aurora B (pAURKB), representing “activated” Aurora B kinase, is diffusely localized during anaphase in both siLUC and siKIF cells. In early midbodies, pAURKB localizes to the center dark zone and inner flanks as a diffuse blob, but appears as a more compact disk in later midbodies, similar in siLUC and siKIF cells. (G, H) Anillin (ANLN) localizes to the furrowing cell cortex during anaphase in siLUC and siKIF cells. In the early midbody stage, ANLN forms a wide ring around the center of the midbody. In late midbodies, ANLN can be found in the center of the midbody as well as at the constriction sites. These localizations were not disrupted in KIF20B-depleted cells. (I, J) α-Actinin-4 (ACTN4) is distributed on the entire cell cortex but clearly accumulates in the cleavage furrow during anaphase in both siLUC and siKIF cells. In early midbody stage, ACTN4 enriches in a half-circle shape at the edges of both daughter cells underneath the midbody. In the late midbody stage, there is no longer enrichment of ACTN4 around the midbody, in both siLUC and siKIF cells. Images in panels A and B and E–J were taken with a DeltaVision deconvolution microscope and those in C and D with a wide-field microscope. At least 15 midbody-stage and five anaphase cells imaged for each marker and condition. Scale bars are 5 µm for all images.

Article Snippet: Primary antibodies used were as follows: mouse monoclonal DM1α (α-tubulin; 1:500) was from Abcam; rat anti-TUBA1A (clone YL ½; 1:750) was from Novus Biologicals; mouse polyclonal anti-CEP55 (1:200) was from Abnova; mouse anti-Aurora kinase B (AURKB; 1:300) was from BD Biosciences; rabbit anti-phospho-T232-Aurora kinase B (pAURKB; 1:200) was from Rockland; rabbit anti-KIF20A (A300-879A; 1:100) was from Bethyl Labs; goat anti-anillin (ANLN; 1:300), mouse monoclonal anti-ANLN (1:100), rabbit anti-MKLP1 (sc-867; 1:100), rabbit anti-PRC1 (1:50), mouse monoclonal anti-human-spastin (3G11/1; 1:50), and mouse anti-human-MPP1(KIF20B; 1:300) were from Santa Cruz; rabbit anti-mouse-Kif20b (1:500) was custom-made by Covance ( Janisch, Vock, et al. , 2013 ); rabbit anti-cleaved-caspase 3 (CC3; 1:200) and rabbit anti-phosphohistone H3 (PH3, Alexa Fluor 647 conjugated; 1:400) were from Cell Signaling; rabbit anti-α-ACTININ4 (ACTN4; 1:250) was from Millipore; and rabbit anti-VPS4 (1:500) was from Sigma Aldrich.

Techniques: Microscopy, Marker

Journal: Molecular Biology of the Cell

Article Title: Kinesin-6 KIF20B is required for efficient cytokinetic furrowing and timely abscission in human cells

doi: 10.1091/mbc.E17-08-0495

Figure Lengend Snippet: Midbodies of KIF20B-depleted cells show reduced frequency of constriction sites and increased CEP55 intensity. (A, B) Representative wide-field images of tubulin in midbodies with one or two constriction sites visible (arrows). (C) The average percentage of midbodies having at least one visible constriction site was reduced in KIF20B-depleted cells at 24 h posttransfection (**: p = 0.01, n = 5 experiments with two coverslips each). (D) Representative deconvolved images of endogenous CEP55 localization to the midbody bulge in both siLUC- and siKIF20B-treated cells. In midbodies without constriction sites, CEP55 appears in two distinct disks. In late midbodies having constriction sites (arrowheads), CEP55 is denser but can still be resolved into two disks by deconvolution in control cells, but not in siKIF-treated cells. (E) The average area of CEP55 signal in the core of late midbodies with constriction sites is not significantly different between siLUC- and siKIF-treated cells. (F) The average maximum fluorescence intensity of CEP55 is significantly higher in late midbodies with constriction sites after KIF20B depletion. **: p = 0.01, n = 26 siLUC and 17 siKIF late midbodies. p Values calculated with a two-tailed Student’s t test, except that in C a paired t test was used. Scale bars for A and B, 5 µm; for D–K, 2.5 µm.

Article Snippet: Primary antibodies used were as follows: mouse monoclonal DM1α (α-tubulin; 1:500) was from Abcam; rat anti-TUBA1A (clone YL ½; 1:750) was from Novus Biologicals; mouse polyclonal anti-CEP55 (1:200) was from Abnova; mouse anti-Aurora kinase B (AURKB; 1:300) was from BD Biosciences; rabbit anti-phospho-T232-Aurora kinase B (pAURKB; 1:200) was from Rockland; rabbit anti-KIF20A (A300-879A; 1:100) was from Bethyl Labs; goat anti-anillin (ANLN; 1:300), mouse monoclonal anti-ANLN (1:100), rabbit anti-MKLP1 (sc-867; 1:100), rabbit anti-PRC1 (1:50), mouse monoclonal anti-human-spastin (3G11/1; 1:50), and mouse anti-human-MPP1(KIF20B; 1:300) were from Santa Cruz; rabbit anti-mouse-Kif20b (1:500) was custom-made by Covance ( Janisch, Vock, et al. , 2013 ); rabbit anti-cleaved-caspase 3 (CC3; 1:200) and rabbit anti-phosphohistone H3 (PH3, Alexa Fluor 647 conjugated; 1:400) were from Cell Signaling; rabbit anti-α-ACTININ4 (ACTN4; 1:250) was from Millipore; and rabbit anti-VPS4 (1:500) was from Sigma Aldrich.

Techniques: Fluorescence, Two Tailed Test

Journal: Molecular Biology of the Cell

Article Title: Kinesin-6 KIF20B is required for efficient cytokinetic furrowing and timely abscission in human cells

doi: 10.1091/mbc.E17-08-0495

Figure Lengend Snippet: Pattern of anillin and VPS4 recruitment is consistent with late-stage maturation defect in KIF20B-depleted midbodies. (A) Left, example deconvolution images of endogenous VPS4 and anillin localization in representative midbodies of HeLa cells. Right, schematic representations of staining in images on left. Arrows point to the central bulge region, and arrowheads point to constriction sites. White, anillin; green, VPS4; red, tubulin. Scale bar, 1 µm. (B) The percentage of midbodies with anillin enriched in the center is increased, and the percentage with VPS4 enriched in the center is decreased. n = 106 siLUC-treated cells, and 109 siKIF-treated cells from two coverslips each of two independent experiments. (C) The percentage of constriction sites (cs) that have anillin enrichment was increased, while the percentage of constriction sites that have VPS4 enrichment was decreased, in KIF20B depleted midbodies, but did not reach statistical significance. n = 48 siLUC constriction sites in 38 midbodies, and n = 46 siKIF constriction sites in 34 midbodies. (D) Bar plot of anillin and VPS4 co-occurrence in midbodies shows KIF20B-depleted midbodies are significantly shifted out of the latest-stage category (VPS4-only) and into the early (anillin-only) and transitional (anillin plus VPS4) categories. n = 106 siLUC-treated midbodies, n = 109 siKIF-treated midbodies. (E) Detailed schematic representations and raw tallies of subcategories of anillin and VPS4 enrichment data plotted in bar graphs in B–D. White space in microtubules symbolizes the central dark zone, and pointed ends symbolize constriction sites (cs). Anillin (blue) or VPS4 (green) enrichment was scored at midbody centers or constriction sites. * p < 0.05; **** p < 0.0001; n.s., not significant (Fisher’s test for B and C, Chi-square test for D).

Article Snippet: Primary antibodies used were as follows: mouse monoclonal DM1α (α-tubulin; 1:500) was from Abcam; rat anti-TUBA1A (clone YL ½; 1:750) was from Novus Biologicals; mouse polyclonal anti-CEP55 (1:200) was from Abnova; mouse anti-Aurora kinase B (AURKB; 1:300) was from BD Biosciences; rabbit anti-phospho-T232-Aurora kinase B (pAURKB; 1:200) was from Rockland; rabbit anti-KIF20A (A300-879A; 1:100) was from Bethyl Labs; goat anti-anillin (ANLN; 1:300), mouse monoclonal anti-ANLN (1:100), rabbit anti-MKLP1 (sc-867; 1:100), rabbit anti-PRC1 (1:50), mouse monoclonal anti-human-spastin (3G11/1; 1:50), and mouse anti-human-MPP1(KIF20B; 1:300) were from Santa Cruz; rabbit anti-mouse-Kif20b (1:500) was custom-made by Covance ( Janisch, Vock, et al. , 2013 ); rabbit anti-cleaved-caspase 3 (CC3; 1:200) and rabbit anti-phosphohistone H3 (PH3, Alexa Fluor 647 conjugated; 1:400) were from Cell Signaling; rabbit anti-α-ACTININ4 (ACTN4; 1:250) was from Millipore; and rabbit anti-VPS4 (1:500) was from Sigma Aldrich.

Techniques: Staining

Journal: Molecular Biology of the Cell

Article Title: Kinesin-6 KIF20B is required for efficient cytokinetic furrowing and timely abscission in human cells

doi: 10.1091/mbc.E17-08-0495

Figure Lengend Snippet: KIF20B depletion delays and dysregulates abscission. (A, B) Example wide-field time-lapse imaging of abscission in a HeLa cell labeled with SiR-tubulin at 20× (A) and 63× (B) (arrowhead a1: abscission 1, arrowhead a2: abscission 2; filled arrow: midbody flanks, open arrow: midbody remnant). Images captured every 7.5 min (A) and every 15 min (B). Insets in B show the intact midbody and the first abscission. (C) Frequency distribution of time from anaphase to first abscission shows a similar median time (67.5 min, arrows, p M-W = 0.13) and similar distribution shape (p K-S = 0.6) for siLUC (black bars) and siKIF-treated cells (white bars) imaged and scored using SiR-tubulin. n = 44 siLUC cells and 41 siKIF20B cells from four experiments. (D) The median time between first and second abscissions (arrows, 7.5 min, p M-W = 0.09) imaged with SiR-tubulin was not different in KIF20B-depleted cells (siKIF) and controls (siLUC). The distributions were not significantly different between siLUC- and sKIF-treated cells (p K-S = 0.9). The sample is the same as in C. (E) Representative selected planes from time-lapse z-stack images of abscission using differential interference contrast (DIC) microscopy, captured every 7.5 min (arrowhead with “a” signifies abscission). Top, in this siLUC-treated cell, abscission was observed 67.5 min after anaphase onset. Bottom, in this siKIF-treated cell, abscission was observed 90 min after anaphase onset. The second abscission event was not discernible by DIC imaging. (F) Increased median time (67.5 vs. 82.5 min) and altered distribution of time to abscission after KIF20B depletion, shown in a frequency graph of time from anaphase to first abscission discerned using DIC time-lapse microscopy in siKIF- (white bars) compared with siLUC-treated cells (black bars). For distribution, * p K-S = 0.04; for medians, * p M-W = 0.04. n = 25 siLUC cells and 30 siKIF cells from four experiments. K-S: Kolmogorov-Smirnov test; M-W: Mann-Whitney U test. Scale bars, 10 µm.

Article Snippet: Primary antibodies used were as follows: mouse monoclonal DM1α (α-tubulin; 1:500) was from Abcam; rat anti-TUBA1A (clone YL ½; 1:750) was from Novus Biologicals; mouse polyclonal anti-CEP55 (1:200) was from Abnova; mouse anti-Aurora kinase B (AURKB; 1:300) was from BD Biosciences; rabbit anti-phospho-T232-Aurora kinase B (pAURKB; 1:200) was from Rockland; rabbit anti-KIF20A (A300-879A; 1:100) was from Bethyl Labs; goat anti-anillin (ANLN; 1:300), mouse monoclonal anti-ANLN (1:100), rabbit anti-MKLP1 (sc-867; 1:100), rabbit anti-PRC1 (1:50), mouse monoclonal anti-human-spastin (3G11/1; 1:50), and mouse anti-human-MPP1(KIF20B; 1:300) were from Santa Cruz; rabbit anti-mouse-Kif20b (1:500) was custom-made by Covance ( Janisch, Vock, et al. , 2013 ); rabbit anti-cleaved-caspase 3 (CC3; 1:200) and rabbit anti-phosphohistone H3 (PH3, Alexa Fluor 647 conjugated; 1:400) were from Cell Signaling; rabbit anti-α-ACTININ4 (ACTN4; 1:250) was from Millipore; and rabbit anti-VPS4 (1:500) was from Sigma Aldrich.

Techniques: Imaging, Labeling, Microscopy, Time-lapse Microscopy, MANN-WHITNEY