rabbit anti p57  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Impaired Myelination in Multiple Sclerosis Organoids: p21 Links Oligodendrocyte Dysfunction to Disease Subtype

doi: 10.1101/2025.01.08.631924

Figure Lengend Snippet: A) Schematic representation of the cell cycle and the involvement of the various CDK/cyclin complexes and the different CDK inhibitors. B) Representative images of an immunofluorescence against the CDKi p16 in c-organoids at D42 in the different conditions. No ectopic location or important change in p16 expression were observed. C) Representative images of an immunofluorescence against the CDKi p57 in c-organoids at D42 in the different conditions. p57 was evenly distributed among the layers of c-organoids. No ectopic location or important change in p57 expression were observed. D) Representative images of an immunofluorescence against the CDKi p21 in c-organoids at D42 in the different conditions. p21 was mostly expressed in the lower layers (SVZ and VZ) of c-organoids. No ectopic location was detected, however, an important decrease of p21 expression was observed, especially in PPMS. E) Representative images of an immunofluorescence against the CDKi p27 in c-organoids at D42 in the different conditions. p27 was mostly expressed in the outer layers of c-organoids (CP). No ectopic location or important change in p27 expression were observed. F) Quantification for p16 (One-Way ANOVA, p = 0.7347), p27 (One-Way ANOVA, p = 0.5313), p57 (One-Way ANOVA, p = 0.7257) and p21 (One-Way ANOVA, p = 0.0004). To ensure quantification consistency, analyzed cortical area were cropped from the original picture to a 250 µm wide×300 µm image, spanning all cortical layers (VZ, SVZ, and CP). Each dot represents one organoid, 4-6 pictures were taken per organoid.

Article Snippet: Slides were then incubated overnight at 4°C with the following primary antibodies diluted in blocking solution: mouse anti-APC (1:100, Calbiochem), mouse anti-ChAT (1:200, ThermoFisher), rat anti-CTIP2 (1:500, Abcam), guinea pig anti-DCX (1:500, Millipore), Rabbit anti-E2F1 (1:400, ThermoFisher), mouse anti-EOMES (1:100, ThermoFisher), mouse anti-GAD67 (1:200, Abcam), mouse anti-GFAP (1:500, Novus Biologicals), mouse anti-Ki67 (1:400, Millipore), chicken anti-MBP (1:500, ThermoFisher), mouse anti-Nanog (1:400, Abcam), mouse anti-O4 (1:200, R&D systems), rabbit anti-Oct4 (1:400, Abcam), rabbit anti-Olig2 (1:200, Abcam), rabbit anti-p16 (1:200, ThermoFisher), rabbit anti-p21 (1:200, ThermoFisher), rabbit anti-p27 (1:400, ThermoFisher), rabbit anti-p57 (1:400, ThermoFisher), mouse anti-Pax6 (1:100, Abcam), rabbit anti-PAK1 (1:100, ThermoFisher), mouse anti-SOX2 (1:400, Abcam), rabbit anti-TBR1 (1:400, Abcam), rabbit anti-vGluT1 (1:200, Abcam).

Techniques: Immunofluorescence, Expressing

Journal: bioRxiv

Article Title: Impaired Myelination in Multiple Sclerosis Organoids: p21 Links Oligodendrocyte Dysfunction to Disease Subtype

doi: 10.1101/2025.01.08.631924

Figure Lengend Snippet: A) Representative images of an immunofluorescence performed on IPS-derived NPC in expansion for p57, p21, p27 and p16, in control and PPMS samples. Only p16 expression was different between the two conditions. B) Representative images of an immunofluorescence performed on IPS-derived NPC, 8 days after mitogens withdrawal for p57 and p27 in control and PPMS samples. p57 and p27 were also co-stained with PAX6 and CTIP2, respectively.

Article Snippet: Slides were then incubated overnight at 4°C with the following primary antibodies diluted in blocking solution: mouse anti-APC (1:100, Calbiochem), mouse anti-ChAT (1:200, ThermoFisher), rat anti-CTIP2 (1:500, Abcam), guinea pig anti-DCX (1:500, Millipore), Rabbit anti-E2F1 (1:400, ThermoFisher), mouse anti-EOMES (1:100, ThermoFisher), mouse anti-GAD67 (1:200, Abcam), mouse anti-GFAP (1:500, Novus Biologicals), mouse anti-Ki67 (1:400, Millipore), chicken anti-MBP (1:500, ThermoFisher), mouse anti-Nanog (1:400, Abcam), mouse anti-O4 (1:200, R&D systems), rabbit anti-Oct4 (1:400, Abcam), rabbit anti-Olig2 (1:200, Abcam), rabbit anti-p16 (1:200, ThermoFisher), rabbit anti-p21 (1:200, ThermoFisher), rabbit anti-p27 (1:400, ThermoFisher), rabbit anti-p57 (1:400, ThermoFisher), mouse anti-Pax6 (1:100, Abcam), rabbit anti-PAK1 (1:100, ThermoFisher), mouse anti-SOX2 (1:400, Abcam), rabbit anti-TBR1 (1:400, Abcam), rabbit anti-vGluT1 (1:200, Abcam).

Techniques: Immunofluorescence, Derivative Assay, Control, Expressing, Staining

Journal: bioRxiv

Article Title: A single cell atlas of mouse podocytes upon injury identifies kidney zone-dependent responses

doi: 10.64898/2026.02.26.708349

Figure Lengend Snippet: (A) Mouse breeding schema showing the crosses of Podocin rtTA, p53 fl/fl and tetO-cre mice used to establish mice with doxycycline inducible podocyte specific ablation of p53. ( B ) Kidney function. Bar Graph of albumin creatinine ratios(ACR) at baseline and day 14 of FSGS in p53 +/+ (light gray) and p53 fl/fl (dark gray) mice. ACR was higher in p53 +/+ compared with p53 fl/fl mice ( C ) Podocyte number . Representative images of p57 staining in the OC and JM and bar graphs of quantification of the number of p57 positive cells per glomerular cross section. Podocyte number was lower in p53 +/+ compared with p53 fl/fl mice ( D ) Glomerulosclerosis. Representative images of Jones silver staining in the OC and JM and bar graphs of quantification of the percentage of the glomerular area stained with silver. Silver staining was higher in p53 +/+ compared with p53 fl/fl mice indicating matrix expansion and more scarring.

Article Snippet: To identify podocyte injury and determine glomerulosclerosis, p57/PAS staining was performed, the following antibody was applied: rabbit anti-p57 (#sc-56341, 1:1000, Santa Cruz Biotechnology).

Techniques: Staining, Silver Staining

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: The EMBO Journal

Article Title: p53 regulates DREAM complex-mediated repression in a p21-independent manner

doi: 10.1038/s44318-025-00402-7

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rabbit anti-p57 (used for WB) , Cell Signaling Technology , Cat# 2557, RRID: AB_2291591 (WB 1:1000).

Techniques: Reverse Transcription, SYBR Green Assay, Extraction, Bicinchoninic Acid Protein Assay, Plasmid Preparation, Mutagenesis, Recombinant, Cloning, Expressing, Control, Sequencing, Software

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