mouse monoclonal anti pml (Santa Cruz Biotechnology)

Santa Cruz Biotechnology mouse monoclonal anti pml

Fig. 3. Recruitment of p53 and CBP to <t>PML</t> NBs. (A and B) Representative images of WI38 normal human fibroblasts infected with B0 (control) or the respective PML-isoforms co-stained with <t>either</t> <t>anti-PML</t> (red) and anti-p53 (green) (A) or anti-CBP (green) (B), and analysed by laser scanning microscopy. The merged image of both stainings is shown in yellow. (C) Forced deposition of p53 into PML NBs. Representative images of WI38 normal human fibroblasts infected with a GFP fusion protein consisting of the minimal PML NB-targeting domain of SP100 and the p53 binding domain of MDM2 (SP–MDM2). SP–MDM2 was localized by direct GFP fluorescence (green) and p53 was visualized with an anti-p53 antibody (red). The merge appears in yellow. Note that SP–MDM2 colocalizes 100% with PML NBs (data not shown).

Mouse Monoclonal Anti Pml, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pg m3 (Santa Cruz Biotechnology)

Santa Cruz Biotechnology pg m3

Localization of PML was examined by indirect immunofluorescence microscopy in mock infected, infected (1.0 or 0.1 PFU/cell HSV-1) and IFN-γ treated MRC5 cells. Cells were fixed 24h post treatment and immunostained with anti-PML <t>(PG-M3)</t> and Texas- Red conjugated secondary antibody. DAPI was used to counter-stain the nucleus.

Pg M3, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pg m3 - by Bioz Stars, 2026-10

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Fig. 3. Recruitment of p53 and CBP to PML NBs. (A and B) Representative images of WI38 normal human fibroblasts infected with B0 (control) or the respective PML-isoforms co-stained with either anti-PML (red) and anti-p53 (green) (A) or anti-CBP (green) (B), and analysed by laser scanning microscopy. The merged image of both stainings is shown in yellow. (C) Forced deposition of p53 into PML NBs. Representative images of WI38 normal human fibroblasts infected with a GFP fusion protein consisting of the minimal PML NB-targeting domain of SP100 and the p53 binding domain of MDM2 (SP–MDM2). SP–MDM2 was localized by direct GFP fluorescence (green) and p53 was visualized with an anti-p53 antibody (red). The merge appears in yellow. Note that SP–MDM2 colocalizes 100% with PML NBs (data not shown).

Journal:

Article Title: Deconstructing PML-induced premature senescence

doi: 10.1093/emboj/cdf341

Figure Lengend Snippet: Fig. 3. Recruitment of p53 and CBP to PML NBs. (A and B) Representative images of WI38 normal human fibroblasts infected with B0 (control) or the respective PML-isoforms co-stained with either anti-PML (red) and anti-p53 (green) (A) or anti-CBP (green) (B), and analysed by laser scanning microscopy. The merged image of both stainings is shown in yellow. (C) Forced deposition of p53 into PML NBs. Representative images of WI38 normal human fibroblasts infected with a GFP fusion protein consisting of the minimal PML NB-targeting domain of SP100 and the p53 binding domain of MDM2 (SP–MDM2). SP–MDM2 was localized by direct GFP fluorescence (green) and p53 was visualized with an anti-p53 antibody (red). The merge appears in yellow. Note that SP–MDM2 colocalizes 100% with PML NBs (data not shown).

Article Snippet: Primary antibodies used were: mouse monoclonal anti-PML (PG-M3; Santa Cruz), mouse monoclonal anti-p53 (DO-1; Santa Cruz), rabbit polyclonal anti-p53 (FL393; Santa Cruz), mouse monoclonal anti-p53 (DO-7, CM-1; Novocastra, UK), mouse monoclonal anti-CBP (C-1; Santa Cruz), rabbit polyclonal anti-GFP (Clontech, MA) and rabbit polyclonal anti-PML (83), as described previously ( Weis et al ., 1994 ).

Techniques: Infection, Staining, Laser-Scanning Microscopy, Binding Assay, Fluorescence

Fig. 4. Sumoylation and intact PML NBs are dispensable for premature senescence. WI38 normal human fibroblasts were infected with B0 (control), PML IV or the SUMO-deficient mutant PML IV-3K retroviruses as described in Figure 2, and growth curves (A), percentage of positive SA-β-Gal-stained cells and %LN (B) were determined. (C) Representative images of WI38 normal human fibroblasts infected with PML IV-3K, co-stained with either anti-PML (red) and anti-p53 (green) or anti-CBP (green), and analysed by laser scanning microscopy. (D–F) WI38 normal human fibroblasts were infected with IE1wt or IE1mt retroviruses and selected for 14 days in 400 µg/ml G418. Cells were then superinfected with either B0 (control), PML IV or oncogenic Ras retroviruses, and selected for 4 days in 3 µg/ml puromycin. (D) Representative images of WI38 normal human fibroblasts double-infected with either IE1wt+PML IV (a), IE1mt+PML IV (b) or IE1wt+Ras and IE1mt+Ras (c) retroviruses. Cells were co-stained either with anti-PML (red) and anti-p53 (green) or anti-CBP (green), and analysed by laser scanning microscopy. (E) Growth curves of WI38 fibroblasts co-infected with the indicated IE1mt+B0, IE1wt+B0, IE1wt+IV or IE1mt+IV, IE1wt+Ras or IE1mt+Ras retroviral constructs. IE1wt/mt+IV, cells transduced first with IE1wt or IE1mt followed by transduction with PML IV. (F) %LN and percentage of positive SA-β-Gal-stained cells from (E) at the indicated time points.

Journal:

Article Title: Deconstructing PML-induced premature senescence

doi: 10.1093/emboj/cdf341

Figure Lengend Snippet: Fig. 4. Sumoylation and intact PML NBs are dispensable for premature senescence. WI38 normal human fibroblasts were infected with B0 (control), PML IV or the SUMO-deficient mutant PML IV-3K retroviruses as described in Figure 2, and growth curves (A), percentage of positive SA-β-Gal-stained cells and %LN (B) were determined. (C) Representative images of WI38 normal human fibroblasts infected with PML IV-3K, co-stained with either anti-PML (red) and anti-p53 (green) or anti-CBP (green), and analysed by laser scanning microscopy. (D–F) WI38 normal human fibroblasts were infected with IE1wt or IE1mt retroviruses and selected for 14 days in 400 µg/ml G418. Cells were then superinfected with either B0 (control), PML IV or oncogenic Ras retroviruses, and selected for 4 days in 3 µg/ml puromycin. (D) Representative images of WI38 normal human fibroblasts double-infected with either IE1wt+PML IV (a), IE1mt+PML IV (b) or IE1wt+Ras and IE1mt+Ras (c) retroviruses. Cells were co-stained either with anti-PML (red) and anti-p53 (green) or anti-CBP (green), and analysed by laser scanning microscopy. (E) Growth curves of WI38 fibroblasts co-infected with the indicated IE1mt+B0, IE1wt+B0, IE1wt+IV or IE1mt+IV, IE1wt+Ras or IE1mt+Ras retroviral constructs. IE1wt/mt+IV, cells transduced first with IE1wt or IE1mt followed by transduction with PML IV. (F) %LN and percentage of positive SA-β-Gal-stained cells from (E) at the indicated time points.

Techniques: Infection, Mutagenesis, Staining, Laser-Scanning Microscopy, Construct, Transduction

Fig. 5. PML IV stabilizes and activates p53. (A) WI38 normal human fibroblasts were infected with either B0 (control), PML III or PML IV retroviruses, as described in the legend to Figure 2. Cells were treated with 10 µg/ml cycloheximide for the indicated times when the PML IV-infected culture was terminally arrested, and whole-cell lysates were prepared and probed for p53. (B) WI38 normal human fibroblasts were infected with either B0 (control), PML I, III, IV, IE1wt or IE1wt+PML IV as described in the legend to Figure 2. Cell lysates were prepared when the PML IV-infected culture was terminally arrested, immunoprecipitated with goat anti-p53 antibody and analysed by western blot using anti-p53-acetylLys382 antibody. (C) Total lysates were prepared from WI38 normal human fibroblasts infected with B0 (control) or the indicated PML retroviruses as in Figure 2, and analysed by western blot using anti-p53-phosphoSer46 antibody and anti-QM antibody for normalization. (D) p53 transactivation activity in quiescent, senescent and PML-infected cells. WI38 fibroblasts were infected with the indicated PML and/or IE1wt retroviruses, and selected and transiently co-transfected with a pCMV-β-galactosidase normalization vector and the p21-Luc reporter when the PML IV-infected culture was completely growth arrested. Cells were assayed for β-galactosidase and luciferase reporters. Normalized p21 reporter activity is luciferase/ β-galactosidase activity, with the activity of p21-Luc in proliferating cells set at 1. Shown are the averages of three independent experiments. Within each experiment, transfections were performed in triplicate. Pre-senescent or senescent cells were cultured in 10% serum, in which case pre-senescent cells were growing exponentially, or incubated in 0.2% serum for 3 days, in which case pre senescent cells were quiescent.

Journal:

Article Title: Deconstructing PML-induced premature senescence

doi: 10.1093/emboj/cdf341

Figure Lengend Snippet: Fig. 5. PML IV stabilizes and activates p53. (A) WI38 normal human fibroblasts were infected with either B0 (control), PML III or PML IV retroviruses, as described in the legend to Figure 2. Cells were treated with 10 µg/ml cycloheximide for the indicated times when the PML IV-infected culture was terminally arrested, and whole-cell lysates were prepared and probed for p53. (B) WI38 normal human fibroblasts were infected with either B0 (control), PML I, III, IV, IE1wt or IE1wt+PML IV as described in the legend to Figure 2. Cell lysates were prepared when the PML IV-infected culture was terminally arrested, immunoprecipitated with goat anti-p53 antibody and analysed by western blot using anti-p53-acetylLys382 antibody. (C) Total lysates were prepared from WI38 normal human fibroblasts infected with B0 (control) or the indicated PML retroviruses as in Figure 2, and analysed by western blot using anti-p53-phosphoSer46 antibody and anti-QM antibody for normalization. (D) p53 transactivation activity in quiescent, senescent and PML-infected cells. WI38 fibroblasts were infected with the indicated PML and/or IE1wt retroviruses, and selected and transiently co-transfected with a pCMV-β-galactosidase normalization vector and the p21-Luc reporter when the PML IV-infected culture was completely growth arrested. Cells were assayed for β-galactosidase and luciferase reporters. Normalized p21 reporter activity is luciferase/ β-galactosidase activity, with the activity of p21-Luc in proliferating cells set at 1. Shown are the averages of three independent experiments. Within each experiment, transfections were performed in triplicate. Pre-senescent or senescent cells were cultured in 10% serum, in which case pre-senescent cells were growing exponentially, or incubated in 0.2% serum for 3 days, in which case pre senescent cells were quiescent.

Techniques: Infection, Immunoprecipitation, Western Blot, Activity Assay, Transfection, Plasmid Preparation, Luciferase, Cell Culture, Incubation

$Fig. 6. PML–/– MEFs are refractory to PML-induced premature senescence. (A) PML–/– MEFs or PML+/+ MEFs were infected with the respective PML retroviruses (in roman numbers) or empty vector control (B0), and growth curves were determined as described in the legend to Figure 2. (B) PML–/– MEFs do not recruit endogenous p53 to neoformed PML NBs. Representative images of PML–/– MEFs or PML+/+ MEFs infected with PML IV retroviruses and co-stained with either anti-PML (red) and anti-p53 (green) or anti-CBP (green), and analysed by laser scanning microscopy. (C) PML–/– MEFs show no increased acetylation of p53 at Lys382 upon PML overexpression. Immunoprecipitation of p53 from cells infected with the indicated retroviruses, and western blot analysis with anti-p53-acetylLys382 antibody as described in the legend to Figure 5.$

Journal:

Article Title: Deconstructing PML-induced premature senescence

doi: 10.1093/emboj/cdf341

Figure Lengend Snippet: Fig. 6. PML–/– MEFs are refractory to PML-induced premature senescence. (A) PML–/– MEFs or PML+/+ MEFs were infected with the respective PML retroviruses (in roman numbers) or empty vector control (B0), and growth curves were determined as described in the legend to Figure 2. (B) PML–/– MEFs do not recruit endogenous p53 to neoformed PML NBs. Representative images of PML–/– MEFs or PML+/+ MEFs infected with PML IV retroviruses and co-stained with either anti-PML (red) and anti-p53 (green) or anti-CBP (green), and analysed by laser scanning microscopy. (C) PML–/– MEFs show no increased acetylation of p53 at Lys382 upon PML overexpression. Immunoprecipitation of p53 from cells infected with the indicated retroviruses, and western blot analysis with anti-p53-acetylLys382 antibody as described in the legend to Figure 5.

Techniques: Infection, Plasmid Preparation, Staining, Laser-Scanning Microscopy, Over Expression, Immunoprecipitation, Western Blot

Journal: PLoS ONE

Article Title: A Role for Cytoplasmic PML in Cellular Resistance to Viral Infection

doi: 10.1371/journal.pone.0002277

Figure Lengend Snippet: Localization of PML was examined by indirect immunofluorescence microscopy in mock infected, infected (1.0 or 0.1 PFU/cell HSV-1) and IFN-γ treated MRC5 cells. Cells were fixed 24h post treatment and immunostained with anti-PML (PG-M3) and Texas- Red conjugated secondary antibody. DAPI was used to counter-stain the nucleus.

Article Snippet: The following antibodies were used: mouse monoclonal anti-PML, PG-M3 (Santa Cruz Biotechnology), mouse monoclonal anti β-Actin (Chemicon), and antibodies to HSV-1 proteins ICP0 (1112) and gB (Goodwin Institute).

Techniques: Immunofluorescence, Microscopy, Infection, Staining

(A) Immunolocalization of PML Ib. PML +/+ PML −/− , and B1041-1 mouse fibroblast cell lines transfected with a human PML Ib/pcDNA3.1 construct were immunostained with PG-M3 to determine the intracellular localization of PML Ib by confocal (PML +/+ ) and indirect (PML −/− and B1041-1) immunofluorescence microscopy. DAPI was used to counter-stain the nucleus. (B) Localization of PML isoforms I, IV, and VI was revealed by immunostain with PG-M3 and Texas Red following the transfection of each cDNA into B1041-1 cells. (C) Co-transfection of PMLIb with nuclear PML isoforms induces a redistribution of PML protein to the cytoplasm: PMLIb+PML I (stable transfectant, left image); PMLIb+PML IV (transient transfection, middle image); PMLIb+PML VI (transient transfection, right image). Cells were immunostained with PG-M3 and Texas red conjugated secondary antibody as above. Note that the cells transiently co-transfected with PML Ib+PML VI (leftmost image) are dual stained with PG-M3 and a murine polyclonal PML antibody and show re-localization of endogenous mouse PML. DAPI was used to counter-stain the nucleus. Localization of PML was examined by indirect immunofluorescence microscopy. (D & E) Interaction between nuclear and cytoplasmic PML isoforms. V5-tagged PML I/ pcDNA 4, myc-tagged PML Ib/pcDNA 3.1 and vector constructs were transiently transfected either alone with vector or in combination, into the HEK293 cell line. 48h post-transfection cells were (D) lysed and subjected to co-immunoprecipitation or (E) fixed and immunostained. (D) V5 and myc antibodies were used for immunopreciptiations while HRP-conjugated V5 and myc antibodies were used for immunoblots. As shown in the bottom right panel, myc-tagged PML Ib co-precipitated with V5-tagged PMLI, whereas transfection of PML I or PML Ib alone did not result in co-immunoprecipitation. Panels on the left (top & bottom) represent immunoblots of the crude lysates (10 µg protein/lane), and are included to show the antibody specificity for the V5-tagged PML I and Myc-tagged PML Ib constructs. V = vector control, I = PML I, Ib = PML Ib, I+Ib = co-transfected PML I & PML Ib. (E) Cells co-transfected with both vectors, PML I-V5+pcDNA 3.1-myc, PML Ib-myc+pcDNA 4-V5-his, or PML I-V5+PML Ib-myc and grown on glass cover slips were single stained with a FITC conjugated V5 antibody (bottom two panels), or the anti-myc antibody, 9E10, and anti mouse Texas Red (top two panels) as indicated in the figure.

Journal: PLoS ONE

Article Title: A Role for Cytoplasmic PML in Cellular Resistance to Viral Infection

doi: 10.1371/journal.pone.0002277

Figure Lengend Snippet: (A) Immunolocalization of PML Ib. PML +/+ PML −/− , and B1041-1 mouse fibroblast cell lines transfected with a human PML Ib/pcDNA3.1 construct were immunostained with PG-M3 to determine the intracellular localization of PML Ib by confocal (PML +/+ ) and indirect (PML −/− and B1041-1) immunofluorescence microscopy. DAPI was used to counter-stain the nucleus. (B) Localization of PML isoforms I, IV, and VI was revealed by immunostain with PG-M3 and Texas Red following the transfection of each cDNA into B1041-1 cells. (C) Co-transfection of PMLIb with nuclear PML isoforms induces a redistribution of PML protein to the cytoplasm: PMLIb+PML I (stable transfectant, left image); PMLIb+PML IV (transient transfection, middle image); PMLIb+PML VI (transient transfection, right image). Cells were immunostained with PG-M3 and Texas red conjugated secondary antibody as above. Note that the cells transiently co-transfected with PML Ib+PML VI (leftmost image) are dual stained with PG-M3 and a murine polyclonal PML antibody and show re-localization of endogenous mouse PML. DAPI was used to counter-stain the nucleus. Localization of PML was examined by indirect immunofluorescence microscopy. (D & E) Interaction between nuclear and cytoplasmic PML isoforms. V5-tagged PML I/ pcDNA 4, myc-tagged PML Ib/pcDNA 3.1 and vector constructs were transiently transfected either alone with vector or in combination, into the HEK293 cell line. 48h post-transfection cells were (D) lysed and subjected to co-immunoprecipitation or (E) fixed and immunostained. (D) V5 and myc antibodies were used for immunopreciptiations while HRP-conjugated V5 and myc antibodies were used for immunoblots. As shown in the bottom right panel, myc-tagged PML Ib co-precipitated with V5-tagged PMLI, whereas transfection of PML I or PML Ib alone did not result in co-immunoprecipitation. Panels on the left (top & bottom) represent immunoblots of the crude lysates (10 µg protein/lane), and are included to show the antibody specificity for the V5-tagged PML I and Myc-tagged PML Ib constructs. V = vector control, I = PML I, Ib = PML Ib, I+Ib = co-transfected PML I & PML Ib. (E) Cells co-transfected with both vectors, PML I-V5+pcDNA 3.1-myc, PML Ib-myc+pcDNA 4-V5-his, or PML I-V5+PML Ib-myc and grown on glass cover slips were single stained with a FITC conjugated V5 antibody (bottom two panels), or the anti-myc antibody, 9E10, and anti mouse Texas Red (top two panels) as indicated in the figure.

Techniques: Transfection, Construct, Immunofluorescence, Microscopy, Staining, Cotransfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Control

(A) A schema of the cellular model and infection protocol. (B) B104-1-1 cells were transfected with vector, PML I, or PML Ib. Stable cell clones were selected. PML I and PML Ib cells were immunostained with PG-M3. (C and D). Stable cell clones were infected with 0.01 PFU/mLHSV-1 -F. Infected cells were harvested at the indicated times for Western blot analysis (C), and analysis of viral tier (D). Expression of PML Ib reduced viral protein production in cells infected with HSV-1(F) (C). Protein lysates (10 µg/sample) were separated by electrophoresis and blotted with antibodies against the HSV-1 viral proteins ICP0 and US11. β-Actin was used as loading control. (Results are representative of multiple experiments.) (D) Viral titers at different time points were determined in triplicate by standard plaque assay using VERO cells.

Journal: PLoS ONE

Article Title: A Role for Cytoplasmic PML in Cellular Resistance to Viral Infection

doi: 10.1371/journal.pone.0002277

Figure Lengend Snippet: (A) A schema of the cellular model and infection protocol. (B) B104-1-1 cells were transfected with vector, PML I, or PML Ib. Stable cell clones were selected. PML I and PML Ib cells were immunostained with PG-M3. (C and D). Stable cell clones were infected with 0.01 PFU/mLHSV-1 -F. Infected cells were harvested at the indicated times for Western blot analysis (C), and analysis of viral tier (D). Expression of PML Ib reduced viral protein production in cells infected with HSV-1(F) (C). Protein lysates (10 µg/sample) were separated by electrophoresis and blotted with antibodies against the HSV-1 viral proteins ICP0 and US11. β-Actin was used as loading control. (Results are representative of multiple experiments.) (D) Viral titers at different time points were determined in triplicate by standard plaque assay using VERO cells.

Techniques: Infection, Transfection, Plasmid Preparation, Stable Transfection, Clone Assay, Western Blot, Expressing, Electrophoresis, Control, Plaque Assay

Journal: PLoS ONE

Article Title: A Role for Cytoplasmic PML in Cellular Resistance to Viral Infection

doi: 10.1371/journal.pone.0002277

Figure Lengend Snippet: (A and B). B104-1-1 cells were stably transfected with PMLIb and exposed to 0.1 PFU/cell HSV-1(F). Cells were fixed 28h post infection. Dual color immunofluorescence staining was performed with PG-M3 (diluted 1∶150) (red), and anti-ICP0 antibody (green). Chromosomal DNA was counterstained with cytox orange. Viral proteins were not detected by the PG-M3 antibody in HSV-1 infected Vector control cells (A), and ICP0 stained uniquely from PML in HSV-1 infected PML I+PML Ib co-transfected cells (compare the heavily infected cell in upper right to the recently infected cell in the lower left corner) (B). (C and D) B104-1-1 stably transfected with PML I (C) or PML Ib (D) were exposed to 0.1 PFU/cell HSV-1 (F). Cells were fixed at 2h and 28h post infection as indicated. Images were captured with a Zeiss 510 LSM confocal microscope. (E) Further analysis using the LSM Image Browser software demonstrates co-localization of PML Ib and ICP0 in the cytoplasm of a PML Ib expressing cell infected for 28 h with 0.1 PFU/cell. Red and green represent PML Ib and ICP0 respectively, while cytox orange staining is represented by blue in both the upper image and intensity profile (bottom). The intensity profile was software generated from the fluorescence intensity values encompassed along the red line (upper image).

Techniques: Stable Transfection, Transfection, Infection, Immunofluorescence, Staining, Plasmid Preparation, Control, Microscopy, Software, Expressing, Generated, Fluorescence

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