Journal: Anatomical record (Hoboken, N.J. : 2007)

Article Title: Maternal hypoxia developmentally programs low podocyte endowment in male, but not female offspring.

doi: 10.1002/ar.24369

Figure Lengend Snippet: FIGURE 1 Confocal microscopy of four whole glomeruli at postnatal Day 21 in male normoxic (a) and male hypoxic (b), female normoxic (c), and female hypoxic (d) mice. P57 immunofluorescence (red) indicates podocyte nuclei. Synaptopodin (green) immunostaining indicates podocyte cytoplasm

Article Snippet: One midhilar slice per kidney then underwent 1 hr of antigen retrieval in Dako Citrate Antigen Retrieval Solution (S1699; Dako, Glostrup, Denmark) on a Dako PT Link Instrument (PT10126; Dako) for 60 min at 98 C. Slices were then incubated for 6 days on an orbital shaker (OM8, Ratek, Australia) at 37 C with two primary antibody solutions for podocyte labeling, namely polyclonal rabbit anti-mouse p57 antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA; sc-8,298; for labeling of podocyte nuclei) and polyclonal goat anti-mouse synaptopodin (1:400, Santa Cruz Biotechnology, Santa Cruz, CA; sc-21537; for labeling of podocyte cytoplasm).

Techniques: Confocal Microscopy, Immunofluorescence, Immunostaining

Journal: Anatomical record (Hoboken, N.J. : 2007)

Article Title: Maternal hypoxia developmentally programs low podocyte endowment in male, but not female offspring.

doi: 10.1002/ar.24369

Figure Lengend Snippet: FIGURE 2 Complete series of confocal optical sections through a whole glomerulus. (a) P57 immunofluorescence (red) indicates podocyte nuclei. (b) P57 and synaptopodin (green) double immunostaining of podocytes

Article Snippet: One midhilar slice per kidney then underwent 1 hr of antigen retrieval in Dako Citrate Antigen Retrieval Solution (S1699; Dako, Glostrup, Denmark) on a Dako PT Link Instrument (PT10126; Dako) for 60 min at 98 C. Slices were then incubated for 6 days on an orbital shaker (OM8, Ratek, Australia) at 37 C with two primary antibody solutions for podocyte labeling, namely polyclonal rabbit anti-mouse p57 antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA; sc-8,298; for labeling of podocyte nuclei) and polyclonal goat anti-mouse synaptopodin (1:400, Santa Cruz Biotechnology, Santa Cruz, CA; sc-21537; for labeling of podocyte cytoplasm).

Techniques: Immunofluorescence, Double Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: Hydatidiform Moles: The Contribution of Ancillary Techniques in Refining Their Histopathological Diagnosis

doi: 10.3390/ijms27010142

Figure Lengend Snippet: p57 immunoexpression. ( a ) p57 immunoexpression in CHM, ×10: negative in villous cytotrophoblast and stromal cells (absent, no maternal genome); ( b ) p57 immunoexpression in PHM, ×10: positive in villous cytotrophoblast and stromal cells (nuclear staining present).

Article Snippet: The following primary antibodies were employed: rabbit polyclonal anti-p57 (clone STJ16100411, dilution 1:300, St John’s Laboratory, London, UK), mouse monoclonal anti-Ki-67 (clone NCL-L-Ki67-MM1, dilution 1:100, Leica Novocastra, Nussloch, Germany), rabbit polyclonal anti-hCG (ab9376, dilution 1:100, Abcam, Cambridge, UK), and mouse monoclonal anti-human E-cadherin (clone NCH-38, ready-to-use, Dako, Glostrup, Denmark).

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: p57 Kip2 Phosphorylation Modulates Its Localization, Stability, and Interactions

doi: 10.3390/ijms252011176

Figure Lengend Snippet: Characterization of the p57 isoform pattern. ( A ) 1D/WB analysis of p57 content in different cell lines, like HeLa ctrl and HeLa treated with 100 nM of Dex for 24 h to increase p57 levels, the neuroblastoma cell lines Lan-5 and SHSY5Y, the trophoblast cell line HTR8/SVneo, and Hek293 and U2OS cells transfected with the hp57-FL plasmid. Loading was verified by β -actin analysis ( B ) 2D/WB of p57 from total protein extracts of HeLa, Lan-5 and HTR8/SVneo cells. The linear range of pH 4–7 was employed for the isoelectrofocusing, since the pI of the FL-hp57 is 5.39. Accordingly, the spot focusing at the reported pH is indicated with number 1 (red arrow). ( C ) The 2D/WB analysis of p57 in HeLa cells treated as indicated. Cells were grown in medium supplemented with 100 nM of Dex for 24 h. Subsequently, Dex-treated cells were incubated with staurosporine (STS), a broad-spectrum kinase inhibitor. Then, cells were collected and lysed. Half of the total protein extract was incubated with λ-PPase and then analyzed by 2D/WB in comparison with the PPase-untreated extract and the extract from DEX-exposed cells. Spots from 1 to 3 were indicated by arrows (red arrow indicates spot 1). Mainly, spots 1 and 3 were maintained after staurosporine treatment, then spot 3 disappeared upon PPase assay. ( D ) The 2D/WB analysis of recombinant hp57-FL (hp57) was produced by the IVTT reaction. Arrows indicate the unmodified form (spot 1, red arrow) and two monophosphorylated isoforms (spots 2 and 3). Recombinant hp57 was immunoprecipitated with a rabbit pAbs anti-p57 to confirm the specificity of the detected p57 signal and then incubated with λ-PPase. The 2D/WB analysis showed only the spot focused at pH 5.39 (in red), corresponding to the unmodified hp57. ( E ) The 2D/WB analysis of p57 isoforms in HeLa cells. hp57 isoforms that focus at the same pH are grouped in boxes. The isoform that focuses at pH 5.39 corresponds to the unmodified form of p57 (UM), while shifting toward the acidic pole (left) indicates forms to which a phosphate group is progressively added. 1P, 2P, 3P, and 4P indicate hp57 isoforms containing, respectively, one, two, three, and four phosphate groups. ( F ) The image shows a detail of panel ( E ). A magnification of hp57 monophosphorylated (1P) forms is shown. Arrows point to five distinct monophosphorylated isoforms detected in HeLa cells.

Article Snippet: The IVTT reaction was diluted in RIPA buffer and recombinant hp57 was immunoprecipitated with a rabbit polyclonal anti-p57 Ab conjugated to protein A/G Agarose beads (Santa Cruz Biotechnology, Inc., Heidelberg, Germany).

Techniques: Transfection, Plasmid Preparation, Incubation, Comparison, Recombinant, Produced, Immunoprecipitation

Journal: International Journal of Molecular Sciences

Article Title: p57 Kip2 Phosphorylation Modulates Its Localization, Stability, and Interactions

doi: 10.3390/ijms252011176

Figure Lengend Snippet: Analysis of p57 isoforms in the cytoplasmic and nuclear compartments. ( A ) The 1D/WB analysis of p57 in nuclear and cytoplasm fractionation of the neuroblastoma cells SH-SY5Y, SK-N-BE, and Lan-5. Total extract of HeLa treated with Dex was used as a reference for the hp57 signal. HDAC1 and LDH were evaluated as markers of nuclear and cytosolic fractions, respectively. ( B ) The 2D/WB of p57 in the nuclear and cytoplasmic fractions of asynchronous Lan-5 cells. Arrows indicate the unmodified (UM) p57 form detected only in the nucleus and the other two monophosphorylated (1P) forms. A box is also added to group the 1P p57 isoforms, in nuclear and cytoplasmic fractions. ( C ) The 2D/WB of the nuclear and cytoplasmic fractions of HeK293 cells transfected with pcDNA3.1 plasmid expressing the C-term (121–316)-p57 fragment. Eight isoforms of the C-term fragment were detected and numbered from the basic to acidic pH. Spot 1 (unmodified C-term fragment) was detected only in the nucleus. The less phosphorylated forms of the fragment were detected in the nucleus (spots 2–4), while the most phosphorylated forms localized in the cytoplasm. ( D ) The 2D/WB of the IVTT reaction of the C-term (121–316)-p57 fragment to confirm that spot 1 corresponds to the unmodified form of the fragment. Only one spot was detected that focused at pH 5.92, corresponding to the theoretical calculated pI of the C-term fragment.

Article Snippet: The IVTT reaction was diluted in RIPA buffer and recombinant hp57 was immunoprecipitated with a rabbit polyclonal anti-p57 Ab conjugated to protein A/G Agarose beads (Santa Cruz Biotechnology, Inc., Heidelberg, Germany).

Techniques: Fractionation, Transfection, Plasmid Preparation, Expressing

Journal: International Journal of Molecular Sciences

Article Title: p57 Kip2 Phosphorylation Modulates Its Localization, Stability, and Interactions

doi: 10.3390/ijms252011176

Figure Lengend Snippet: Analysis of p57 isoforms in synchronized cells and evaluation of the binding with CDKs. ( A ) 1D/WB analysis of p57 content in nuclear and cytoplasm fractionation of synchronized Lan-5 neuroblastoma cells obtained through the reported treatment: starvation (STARV) for enrichment in G0/G1; addition of 10% FBS to starved cells (+10% FBS for 8 and 12 h) for cells entering G1 and proceeding toward the S phase; thymidine block for the S phase arrest (dT block); harvesting of attached and shaking-off detached cells after nocodazole treatment (for the G2 and M phase, respectively). Total extract from unsynchronized (GROWING) cells was used as a reference. Western blotting of Lamin A/C and LDH was employed as a marker of, respectively, nuclear and cytoplasmic fractions. ( B ) Graphical representation of FACS analysis results related to Lan-5 cell synchronizations performed as described in the Material and Methods. ( C ) The 2D/WB analysis of hp57 in the nuclear and cytoplasmic fractions of synchronized Lan-5 cells shows the distribution pattern of p57 phosphoisoforms in cell cycle phases. As in E, hp57 isoforms that focus at the same pH are grouped in boxes. The unmodified (UM) and the mono- and biphosphorylated forms (1P and 2P) of hp57 forms are detected in the nucleus through cell cycle phases, while the most phosphorylated hp57 forms (from 1P to 4P) are detected in the cytoplasm in each analyzed phase of the cell cycle (except the S phase). In the G2/M phase, it is evident that hp57 is highly phosphorylated. ( D ) The 1D/WB of cytoplasmic and nuclear fractionation of Lan-5 arrested in the S phase after 48 h of incubation with 2 mM deoxythymidine (dT block). A total of 10 µM of MG132 was added in the last 5 h of dT incubation to prevent proteasome-dependent protein degradation. As in panel A, Western blotting of Lamin A/C and LDH were employed as a marker of nuclear and cytoplasmic fractions, respectively. ( E ) The 2D/WB analysis of hp57 in total Lan-5 extracts arrested in the S phase treated or not with MG132, as in panel (D). ( F ) The 2D/WB analysis of p57 isoforms involved in the binding with CDK4, CDK6, CDK2, and CDK1. A total of 5 mg of Lan-5 extract was used for immunoprecipitation of each CDK. Images acquired at different blot exposure times (1 min and 5 min of exposure) are reported. Boxes highlight the main p57 forms involved in the binding.

Article Snippet: The IVTT reaction was diluted in RIPA buffer and recombinant hp57 was immunoprecipitated with a rabbit polyclonal anti-p57 Ab conjugated to protein A/G Agarose beads (Santa Cruz Biotechnology, Inc., Heidelberg, Germany).

Techniques: Binding Assay, Fractionation, Blocking Assay, Western Blot, Marker, Incubation, Immunoprecipitation

Journal: International Journal of Molecular Sciences

Article Title: p57 Kip2 Phosphorylation Modulates Its Localization, Stability, and Interactions

doi: 10.3390/ijms252011176

Figure Lengend Snippet: Analysis of p57 isoforms in the association with LIMK1 and CRM1. ( A ) The 1D/WB analysis of p57 content in the immunoprecipitation of LIMK1 from total protein extracts of Hek293 cells co-transfected for 24 h with 1 µg of both p57-FL pcdna3.1 plasmid and Myc-DDK-hLIMK1 pCMV6-Entry plasmid. Hek293 cells transfected with an empty vector were used as a negative control of transfection, and total extract is loaded as a reference for LIMK1 and p57 endogenous signals. Hek293 cells transfected with only Myc-DDK-hLIMK1 pCMV6-Entry plasmid were used as a negative control for the IP experiment. Immunoprecipitation (IP) with a rabbit pAb anti-LIMK1 was performed from both cells co-transfected and transfected only with the Myc-DDK-tagged hLIMK1 plasmid. Total extract (INPUT), LIMK1 IP, and supernatants (S/N) after LIMK1 IP were analyzed for p57 and LIMK1 content in 1D/WB. * in red indicates IgG heavy chain’s signal. ( B ) The 1D/WB analysis of the p57 N-term and C-term fragment content in the immunoprecipitation of Myc-DDK-LIMK1 from total protein extracts of Hek293 cells co-transfected as in panel A except that N-term (1–219)-hp57 and the C-term (121–316)-hp57 pcDNA3.1 plasmids were employed. Immunoprecipitation with a mouse anti-Myc mAb was performed from all three transfected conditions. Total extracts and Myc-LIMK1 IP were analyzed for p57 and Myc-tagged LIMK1 content in 1D/WB. A mouse monoclonal and a rabbit polyclonal Ab were employed to detect, respectively, the N-term (1–219)-hp57 and C-term (121–316)-hp57 fragments. Images acquired at different blot exposure times (1 min and 10 min of exposure) are reported. ( C ) The 1D/WB analysis of p57 content in the immunoprecipitation of CRM1 from total protein extracts of Hek293 cells co-transfected as in panels ( A , B ) except that a CRM1 pcDNA3.1 plasmid was employed. Immunoprecipitation with a mouse anti-CRM1 mAb was performed from all four transfected conditions. Total extracts and CRM1 IP were analyzed for p57 and CRM1 content in 1D/WB. A mouse monoclonal anti-p57 Ab was used to detect the FL-p57 and N-term (1–219)-hp57 fragment. A rabbit polyclonal anti-p57 Ab was employed to detect the C-term (121–316)-hp57 fragment. * indicates IgG heavy chain’s signal. ( D ) The 2D/WB analysis of p57 content in the immunoprecipitated material from Myc-LIMK1 and CRM1 immunoprecipitations. Boxes highlight the differences in p57 isoforms involved in the binding with the two interactors analyzed. Specifically, the unmodified (UM) and the monophosphorylated (1P) forms are found in the binding with CRM1, while more acidic (2P and 3P) isoforms are prevalently found in the binding with LIMK1. Images acquired at different blot exposure times (1 min and 10 min of exposure) are reported.

Article Snippet: The IVTT reaction was diluted in RIPA buffer and recombinant hp57 was immunoprecipitated with a rabbit polyclonal anti-p57 Ab conjugated to protein A/G Agarose beads (Santa Cruz Biotechnology, Inc., Heidelberg, Germany).

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Negative Control, Binding Assay

Journal: Redox biology

Article Title: Coronin 1a-mediated F-actin disassembly controls effector function in murine neutrophils.

doi: 10.1016/j.redox.2025.103618

Figure Lengend Snippet: Fig. 2. F-actin disassembly promotes ROS production in neutrophils (A) Cellular F-actin content detected by intracellular phalloidin staining and flow cytometric analysis. Left: representative FACS histogram; Right: relative F-actin level (n = 5). Unpaired Student’s t-test. (B) Confocal microscopy of WT and Coro1a−/−(KO) neutrophils showing staining for F-actin (phalloidin) and Coro1a. Nuclear staining: DAPI. Scale bar, 3 μm. (C–E) PMA (100 ng/ml)-induced ROS production in WT and Coro1a−/−(KO) neutrophils pretreated for 60 min with the indicated concentrations of (C) jasplakinolide (Jas; actin-stabilizing agent), (D) latrunculin B (Lat; actin-depolymerizing agent); or (E) benproperine phosphate (Benp; Arp2/3-complex inhibitor). ROS production was measured with luminol chemiluminescence. Left: ROS response kinetics; Right: normalized integrated ROS signals. Data are mean ± SEM (n = 2). (C–E) 2-way ANOVA test (Tukey). ns > 0.05, ** <0.01, *** <0.001, **** <0.0001. Data are representative for at least 3 independent experiments.

Article Snippet: Co-IP was conducted by incubation of cell lysates with magnetic beads conjugated to either rabbit polyclonal anti-Coro1a antibody (Proteintech) or isotype matched control antibody in an orbital rotator Intelli-Mixer RM-2M for 2 h at 4 ◦C.

Techniques: Staining, Confocal Microscopy

Journal: Redox biology

Article Title: Coronin 1a-mediated F-actin disassembly controls effector function in murine neutrophils.

doi: 10.1016/j.redox.2025.103618

Figure Lengend Snippet: Fig. 3. Enhanced F-actin levels in Coro1a-deficient neutrophils are associated with impaired granule release (A-E) Granule exocytosis (degranulation) by WT and Coro1a−/−(KO) neutrophils. (A, B) Release of myeloperoxidase (MPO) from primary granules into culture supernatants upon stimulation of neutrophils with (A) serum-opsonized whole glucan particles (WGP) or (B) serum-opsonized C. albicans hyphae (SO–CaH). (C) Upregulation of CD63 (Lamp3) cell surface expression upon stimulation with WGP. (D, E) Release of (D) neutrophil gelatinase-associated lipocalin (NGAL) from secondary granules or (E) matrix metalloproteinase-9 (MMP-9) from tertiary granules into culture supernatants upon stimulation of neutrophils with TNFα. (F) Total amount of MPO, NGAL, and MMP-9 in WT and Coro1a−/−(KO) neutrophils expressed in % of WT (n = 5). Unpaired Student’s t-test. (G–I) PMA (100 ng/ml)-induced release of MMP-9 by WT and Coro1a−/−(KO) neutrophils pretreated for 60 min with (G) 16 μM jasplakinolide (Jas; actin-stabilizing agent), (H) 1 μM latrunculin B (Lat; actin-depolymerizing agent); or (I) 30 μM benproperine phosphate (Benp; Arp2/3-complex inhibitor). Data are mean ± SEM (A, D-E: n = 3; B, G-H: n = 2; C: n = 7). (A-E, G-H) 2-way ANOVA. ns > 0.05, * <0.05, ** <0.01, *** <0.001, **** <0.0001. Data are representative for at least 3 independent experiments.

Article Snippet: Co-IP was conducted by incubation of cell lysates with magnetic beads conjugated to either rabbit polyclonal anti-Coro1a antibody (Proteintech) or isotype matched control antibody in an orbital rotator Intelli-Mixer RM-2M for 2 h at 4 ◦C.

Techniques: Expressing

Journal: Redox biology

Article Title: Coronin 1a-mediated F-actin disassembly controls effector function in murine neutrophils.

doi: 10.1016/j.redox.2025.103618

Figure Lengend Snippet: Fig. 4. Local F-actin destruction is required for neutrophil granule exocytosis (A) Transmission electron microscopy (TEM) images of WT and Coro1a−/−(KO) neutrophils either unstimulated or challenged with TNFα (20 ng/ml; 30 min). Micrograph labels: primary (1◦G), secondary – quaternary (2◦G – 4◦G) granules, mitochondria (Mt). TNFα treated condition: non-primary granules are labeled with yellow asterisks. Scale bar, 2 μm; each micrograph is 4.5 μm × 4.5 μm, x22000 magnification. Images shown are representative of at least n = 50 neutrophils being analyzed from each condition. (B) Airyscan-enhanced confocal micrographs of WT and Coro1a−/−(KO) neutrophils in basal state (left panel) or stimulated with 20 ng/ml of TNFα for 30 min (right panel). Neutrophils were fixed permeabilized and stained for F-actin (phalloidin) and MMP-9 together with DAPI (nucleus). Scale bar: 3 μm. Images shown are representative of neutrophils observed in 3 independent microscopy experiments. Each experiment consisted of analysis of 8–12 fields for every condition, with each field containing ca. 10–30 cells.

Article Snippet: Co-IP was conducted by incubation of cell lysates with magnetic beads conjugated to either rabbit polyclonal anti-Coro1a antibody (Proteintech) or isotype matched control antibody in an orbital rotator Intelli-Mixer RM-2M for 2 h at 4 ◦C.

Techniques: Transmission Assay, Electron Microscopy, Labeling, Staining, Microscopy

Journal: Redox biology

Article Title: Coronin 1a-mediated F-actin disassembly controls effector function in murine neutrophils.

doi: 10.1016/j.redox.2025.103618

Figure Lengend Snippet: Fig. 5. Identification of Coro1a-associated protein complexes in neutrophils (A) (Top left) Total scatter plot from co-immunoprecipitation with mass-spectrometry (Co-IP/MS) assay displaying proteins found co-precipitating with Coro1a in 2 biological replicates (WT-1 and WT-2 IP) of neutrophil lysates (plot was generated in Perseus software). (Top right) Magnification of the upper right quadrant of the scatter plot showing the most abundant proteins in the Coro1a-precipitates. (Bottom panels) Selected proteins from the Coro1a-precipitates are divided and color- coded into three groups (green: Ras proteins/NADPH oxidase; blue: neutrophil intragranular compounds (and granule subtype: primary – tertiary (1◦–3◦) granules); magenta: cytoskeletal proteins. IQGAP1, IQ motif-containing GTPase activating protein 1; Ncf4, neutrophil cytosolic factor 4 (alias p40phox); Wdr1, WD-repeat protein (alias AIP1, actin-interacting protein 1); Arp, actin-related protein; Arpc, Arp 2/3 complex proteins. (B, C) WT and Coro1a−/− (KO) neutrophils were stimulated with PMA (100 ng/ml) for the indicated times. Cellular lysates were subjected to Coro1a-immunoprecipitation (IP) followed by Western blot analysis with the indicated Abs. (p-Ser (PKC): phospho-Serine (PKC substrate)). H/C: Ig heavy chain of immunoprecipitating Ab.

Article Snippet: Co-IP was conducted by incubation of cell lysates with magnetic beads conjugated to either rabbit polyclonal anti-Coro1a antibody (Proteintech) or isotype matched control antibody in an orbital rotator Intelli-Mixer RM-2M for 2 h at 4 ◦C.

Techniques: Immunoprecipitation, Mass Spectrometry, Co-Immunoprecipitation Assay, Generated, Software, Western Blot

Journal: Redox biology

Article Title: Coronin 1a-mediated F-actin disassembly controls effector function in murine neutrophils.

doi: 10.1016/j.redox.2025.103618

Figure Lengend Snippet: Fig. 6. Coro1a-mediated activation and membrane translocation of Rac2 controls neutrophil ROS production (A) WT and Coro1a−/−(KO) neutrophils stimulated with PMA (100 ng/ml) for the indicated times were analyzed for Rac1 and Rac2 activation by pull-down assay. Cell extracts were also analyzed for total amounts of Rac1 and Rac2. (B) PMA (100 ng/ml; 10 min)-induced translocation of Rac2 from the cytosol to the membrane was analyzed by subcellular fractionation assay. Gp91phox and HSP70 were used as loading controls for membrane and cytosolic fractions, respectively. (C, D) PMA (100 ng/ml)-induced ROS production in WT and Coro1a−/−(KO) neutrophils pretreated for 60 min with the indicated concentrations of (C) NSC23766 (Rac in hibitor), or (D) ML-099 (pan Ras activator). ROS production was measured with luminol chemiluminescence. Left: ROS response kinetics; Right: normalized inte grated ROS signals. (C, D) Data are mean ± SEM (n = 2). 2-way ANOVA test (Tukey). ns > 0.05, ** <0.01, *** <0.001, **** <0.0001. Data are representative for at least 3 independent experiments.

Article Snippet: Co-IP was conducted by incubation of cell lysates with magnetic beads conjugated to either rabbit polyclonal anti-Coro1a antibody (Proteintech) or isotype matched control antibody in an orbital rotator Intelli-Mixer RM-2M for 2 h at 4 ◦C.

Techniques: Activation Assay, Membrane, Translocation Assay, Pull Down Assay, Fractionation