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Santa Cruz Biotechnology rabbit polyclonal anti p57 ab conjugated to protein a g agarose beads

Characterization of the <t>p57</t> 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.

Rabbit Polyclonal Anti P57 Ab Conjugated To Protein A G Agarose Beads, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 320 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit polyclonal anti p57 ab conjugated to protein a g agarose beads - by Bioz Stars, 2026-08

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1) Product Images from "p57 Kip2 Phosphorylation Modulates Its Localization, Stability, and Interactions"

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

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms252011176

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.

Figure Legend 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.

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

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

Techniques Used: Fractionation, Transfection, Plasmid Preparation, Expressing

$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.$
Figure Legend 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.

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

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.

Figure Legend 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.

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

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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).

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.

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.

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.

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

Journal: iScience

Article Title: Uterine-specific Ezh2 deletion enhances stromal cell senescence and impairs placentation, resulting in pregnancy loss

doi: 10.1016/j.isci.2023.107028

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-p57 , Santa Cruz Biotechnology , Cat# sc-8298; RRID: AB_2078155.

Techniques: Recombinant, Blocking Assay, Plasmid Preparation, Electron Microscopy, Protease Inhibitor, Staining, Bicinchoninic Acid Protein Assay, Software

Cell cycle regulators are affected by the delivery mode, and expressed in trophoblast organoids and placental tissues. ( A ) The relative amount of the gene levels of CDKN1A (p21), CDKN1B (p27), and CDKN1C (p57) was analyzed in placental tissues from different delivery modes ( n = 5). The results are presented as relative quantification (RQ) with minimum and maximum range. The mean value of the expression levels of SDHA (succinate dehydrogenase complex, subunit A), TBP (TATA box–binding protein), and YWHAZ (tyrosine 3–monooxygenase/tryptophan 5–monooxygenase activation protein, zeta polypeptide) served as the endogenous control. CS, caesarean section; eCS, emergency caesarean section after the onset of labor; VD, vaginal delivery; opVD, operative vaginal delivery. Unpaired Student’s t -test was used for statistical analysis, ** p < 0.01. ( B ) Representative immunohistochemistry-immunofluorescence (IHC–IF) image of a trophoblast organoid (8 weeks of gestation) stained for cytokeratin 7 (green), p21 (red), and nuclei (DAPI, blue) is shown. Scale: 50 µm. Inset scale: 10 µm. Villous cytotrophoblasts (CTBs) and the syncytiotrophoblast (STB) are indicated. ( C ) Representative IHC-IF image of placental tissue (8 weeks of gestation) stained for cytokeratin 7 (green), p21 (red), and nuclei (DAPI, blue) is presented. Scale: 10 µm. ( D ) Representative IHC–IF image of placental tissue (7 weeks of gestation) stained for E-cadherin (green), p21 (red), and nuclei (DAPI, blue) is shown. Scale: 10 µm. Inset scale: 5 µm. CTBs ongoing to fuse to the STB (fCTBs), CTBs and the STB are indicated. ( E ) Formalin-fixed and paraffin-embedded (FFPE) tissue sections were immunohistochemically stained with p21 (first row), p–p21 (second row), p27 (third row) or p57 (fourth row) antibody (brown), respectively, and counterstained with hematoxylin (blue). Scale: 50 µm. Inset scale: 20 µm. EVTs (extravillous cytotrophoblasts) are indicated.

Journal: Cells

Article Title: Functional Analysis of p21 Cip1/ CDKN1A and Its Family Members in Trophoblastic Cells of the Placenta and Its Roles in Preeclampsia

doi: 10.3390/cells10092214

Figure Lengend Snippet: Cell cycle regulators are affected by the delivery mode, and expressed in trophoblast organoids and placental tissues. ( A ) The relative amount of the gene levels of CDKN1A (p21), CDKN1B (p27), and CDKN1C (p57) was analyzed in placental tissues from different delivery modes ( n = 5). The results are presented as relative quantification (RQ) with minimum and maximum range. The mean value of the expression levels of SDHA (succinate dehydrogenase complex, subunit A), TBP (TATA box–binding protein), and YWHAZ (tyrosine 3–monooxygenase/tryptophan 5–monooxygenase activation protein, zeta polypeptide) served as the endogenous control. CS, caesarean section; eCS, emergency caesarean section after the onset of labor; VD, vaginal delivery; opVD, operative vaginal delivery. Unpaired Student’s t -test was used for statistical analysis, ** p < 0.01. ( B ) Representative immunohistochemistry-immunofluorescence (IHC–IF) image of a trophoblast organoid (8 weeks of gestation) stained for cytokeratin 7 (green), p21 (red), and nuclei (DAPI, blue) is shown. Scale: 50 µm. Inset scale: 10 µm. Villous cytotrophoblasts (CTBs) and the syncytiotrophoblast (STB) are indicated. ( C ) Representative IHC-IF image of placental tissue (8 weeks of gestation) stained for cytokeratin 7 (green), p21 (red), and nuclei (DAPI, blue) is presented. Scale: 10 µm. ( D ) Representative IHC–IF image of placental tissue (7 weeks of gestation) stained for E-cadherin (green), p21 (red), and nuclei (DAPI, blue) is shown. Scale: 10 µm. Inset scale: 5 µm. CTBs ongoing to fuse to the STB (fCTBs), CTBs and the STB are indicated. ( E ) Formalin-fixed and paraffin-embedded (FFPE) tissue sections were immunohistochemically stained with p21 (first row), p–p21 (second row), p27 (third row) or p57 (fourth row) antibody (brown), respectively, and counterstained with hematoxylin (blue). Scale: 50 µm. Inset scale: 20 µm. EVTs (extravillous cytotrophoblasts) are indicated.

Article Snippet: The following antibodies were used: mouse monoclonal antibody against GAPDH (GTX627408; GeneTex, Eching, Germany) and p53 (DO-1, sc-126; Santa Cruz, Heidelberg, Germany); rabbit monoclonal antibody against p21 (#2947) and p27 (#3686); and rabbit polyclonal antibody against p57 (#2557; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Quantitative Proteomics, Expressing, Binding Assay, Activation Assay, Control, Immunohistochemistry, Immunofluorescence, Immunohistochemistry-IF, Staining

Cell cycle regulators are highly expressed during the first trimester of gestation. ( A – C ) Evaluation of positive cells in first trimester placental sections ( n = 6; named 6–9 week), early-onset control ( n = 20; named 25–33 week), and late-onset control samples ( n = 10; named 34–40 week). The results are presented as bar and scatter plots showing the mean value ± SD. ( A ) Quantification of p21 (left panel), p–p21 (middle panel), and p27 (right panel) positive CTBs in %. ( B ) Quantification of p21 (left panel), p-p21 (middle panel), and p27 (right panel) positive stained STB area in %. ( C ) Quantification of p21 (left panel), p–p21 (second panel), p27 (third panel), and p57 (right panel) positive fCTBs in %. ( D ) The relative amount of the gene levels was analyzed from placental tissues: left panel CDKN1A (p21), middle panel CDKN1B (p27), and right panel CDKN1C (p57). The results are presented as relative quantification (RQ) with minimum and maximum range. TBP was used as the endogenous control. Unpaired Student’s t -test or Mann–Whitney U test referring to first trimester samples was used for statistical analysis, * p < 0.05, ** p < 0.01, *** p < 0.001. CTBs, cytotrophoblasts; fCTBs, cytotrophoblasts ongoing to fuse; STB, syncytiotrophoblast.

Journal: Cells

Article Title: Functional Analysis of p21 Cip1/ CDKN1A and Its Family Members in Trophoblastic Cells of the Placenta and Its Roles in Preeclampsia

doi: 10.3390/cells10092214

Figure Lengend Snippet: Cell cycle regulators are highly expressed during the first trimester of gestation. ( A – C ) Evaluation of positive cells in first trimester placental sections ( n = 6; named 6–9 week), early-onset control ( n = 20; named 25–33 week), and late-onset control samples ( n = 10; named 34–40 week). The results are presented as bar and scatter plots showing the mean value ± SD. ( A ) Quantification of p21 (left panel), p–p21 (middle panel), and p27 (right panel) positive CTBs in %. ( B ) Quantification of p21 (left panel), p-p21 (middle panel), and p27 (right panel) positive stained STB area in %. ( C ) Quantification of p21 (left panel), p–p21 (second panel), p27 (third panel), and p57 (right panel) positive fCTBs in %. ( D ) The relative amount of the gene levels was analyzed from placental tissues: left panel CDKN1A (p21), middle panel CDKN1B (p27), and right panel CDKN1C (p57). The results are presented as relative quantification (RQ) with minimum and maximum range. TBP was used as the endogenous control. Unpaired Student’s t -test or Mann–Whitney U test referring to first trimester samples was used for statistical analysis, * p < 0.05, ** p < 0.01, *** p < 0.001. CTBs, cytotrophoblasts; fCTBs, cytotrophoblasts ongoing to fuse; STB, syncytiotrophoblast.

Techniques: Control, Staining, Quantitative Proteomics, MANN-WHITNEY

p21 expression is reduced in fCTBs of early-onset PE samples. ( A – C ) Quantification of cell cycle regulators in placental sections of control donors (control, n = 20) and patients with early-onset PE (ePE, n = 20) using the H-score method. The results are presented as bar and scatter plots showing the mean value with SD. The percentage of positive stained cells/area is shown under each graph. ( A ) H-score of p21 (left panel), p–p21 (middle panel), and p27 (right panel) for CTBs. ( B ) H-score of p21 (left panel), p–p21 (middle panel), and p27 (right panel) for the STB area. ( C ) H-score of p21 (left panel), p–p21 (second panel), p27 (third panel), and p57 (right panel) for fCTBs. ( D ) Quantification of p21 positive (left panel), p–p21 positive (second panel), p27 positive (third panel), and p57 positive EVTs (right panel) in %. ( E ) Western blot analysis with extracts from placental tissues is shown. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the loading control. ( F ) The relative amount of the gene levels of CDKN1A (p21), CDKN1B (p27), and CDKN1C (p57) was analyzed from placental tissues. The results are presented as relative quantification (RQ) with minimum and maximum range. TBP was used as the endogenous control. Paired Student’s t -test or Wilcoxon-test was used for statistical analysis. CTBs, cytotrophoblasts; fCTBs, cytotrophoblasts ongoing to fuse; STB, syncytiotrophoblast; EVT, extravillous cytotrophoblasts; no, number; MV, mean value.

Journal: Cells

Article Title: Functional Analysis of p21 Cip1/ CDKN1A and Its Family Members in Trophoblastic Cells of the Placenta and Its Roles in Preeclampsia

doi: 10.3390/cells10092214

Figure Lengend Snippet: p21 expression is reduced in fCTBs of early-onset PE samples. ( A – C ) Quantification of cell cycle regulators in placental sections of control donors (control, n = 20) and patients with early-onset PE (ePE, n = 20) using the H-score method. The results are presented as bar and scatter plots showing the mean value with SD. The percentage of positive stained cells/area is shown under each graph. ( A ) H-score of p21 (left panel), p–p21 (middle panel), and p27 (right panel) for CTBs. ( B ) H-score of p21 (left panel), p–p21 (middle panel), and p27 (right panel) for the STB area. ( C ) H-score of p21 (left panel), p–p21 (second panel), p27 (third panel), and p57 (right panel) for fCTBs. ( D ) Quantification of p21 positive (left panel), p–p21 positive (second panel), p27 positive (third panel), and p57 positive EVTs (right panel) in %. ( E ) Western blot analysis with extracts from placental tissues is shown. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the loading control. ( F ) The relative amount of the gene levels of CDKN1A (p21), CDKN1B (p27), and CDKN1C (p57) was analyzed from placental tissues. The results are presented as relative quantification (RQ) with minimum and maximum range. TBP was used as the endogenous control. Paired Student’s t -test or Wilcoxon-test was used for statistical analysis. CTBs, cytotrophoblasts; fCTBs, cytotrophoblasts ongoing to fuse; STB, syncytiotrophoblast; EVT, extravillous cytotrophoblasts; no, number; MV, mean value.

Techniques: Expressing, Control, Staining, Western Blot, Quantitative Proteomics

p21 expression is reduced in early-onset PE complicated by the HELLP syndrome. ( A – C ) Quantification of cell cycle regulators in placental sections of control donors (control, n = 17) and placental tissues from early-onset PE complicated by the HELLP syndrome (ePEH, n = 16) using the H-score method. The results are presented as bar and scatter plots showing the mean value with SD. The percentage of positive stained cells/area is shown under each graph. ( A ) H-score of p21 (left panel), p–p21 (middle panel), and p27 (right panel) for CTBs. ( B ) H-score of p21 (left panel), p–p21 (middle panel), and p27 (right panel) for the STB area. ( C ) H-score of p21 (left panel), p–p21 (second panel), p27 (third panel), and p57 (right panel) for fCTBs. ( D ) Quantification of p21 positive (left panel), p–p21 positive (second panel), p27 positive (third panel), and p57 positive EVTs (right panel) in %. ( E ) Western blot analysis with extracts from placental tissues is shown. GAPDH served as the loading control. ( F ) The relative amount of the gene levels of CDKN1A (p21), CDKN1B (p27) and CDKN1C (p57) was analyzed with placental tissues. The results are presented as relative quantification (RQ) with minimum and maximum range. TBP was used as the endogenous control. Paired Student’s t -test or Wilcoxon-test was used for statistical analysis. CTBs, cytotrophoblasts; fCTBs, cytotrophoblasts ongoing to fuse; STB, syncytiotrophoblast; EVT, extravillous cytotrophoblasts; no, number; MV, mean value.

Journal: Cells

Article Title: Functional Analysis of p21 Cip1/ CDKN1A and Its Family Members in Trophoblastic Cells of the Placenta and Its Roles in Preeclampsia

doi: 10.3390/cells10092214

Figure Lengend Snippet: p21 expression is reduced in early-onset PE complicated by the HELLP syndrome. ( A – C ) Quantification of cell cycle regulators in placental sections of control donors (control, n = 17) and placental tissues from early-onset PE complicated by the HELLP syndrome (ePEH, n = 16) using the H-score method. The results are presented as bar and scatter plots showing the mean value with SD. The percentage of positive stained cells/area is shown under each graph. ( A ) H-score of p21 (left panel), p–p21 (middle panel), and p27 (right panel) for CTBs. ( B ) H-score of p21 (left panel), p–p21 (middle panel), and p27 (right panel) for the STB area. ( C ) H-score of p21 (left panel), p–p21 (second panel), p27 (third panel), and p57 (right panel) for fCTBs. ( D ) Quantification of p21 positive (left panel), p–p21 positive (second panel), p27 positive (third panel), and p57 positive EVTs (right panel) in %. ( E ) Western blot analysis with extracts from placental tissues is shown. GAPDH served as the loading control. ( F ) The relative amount of the gene levels of CDKN1A (p21), CDKN1B (p27) and CDKN1C (p57) was analyzed with placental tissues. The results are presented as relative quantification (RQ) with minimum and maximum range. TBP was used as the endogenous control. Paired Student’s t -test or Wilcoxon-test was used for statistical analysis. CTBs, cytotrophoblasts; fCTBs, cytotrophoblasts ongoing to fuse; STB, syncytiotrophoblast; EVT, extravillous cytotrophoblasts; no, number; MV, mean value.

Techniques: Expressing, Control, Staining, Western Blot, Quantitative Proteomics

p21 expression is decreased in trophoblastic cell lines and in isolated primary trophoblasts under hypoxic conditions. Cells were grown under normoxia (norm, 21.4% O 2 ) or hypoxia (hypox, 1% O 2 ) for 48 h prior to RNA extraction or western blot analysis. ( A ) SGHPL-4 cells. Gene analysis of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57) and TP53 (p53) is shown. The results ( n = 3) are presented as relative quantification (RQ) with minimum and maximum range. GAPDH was used as the endogenous control. ( B ) Western blot analysis with cellular lysates from SGHPL-4 cells ( n = 3). GAPDH served as the loading control. ( C ) HTR cells. Gene analysis of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57), and TP53 (p53) is shown. The results ( n = 3) are presented as relative quantification (RQ) with minimum and maximum range. GAPDH was used as the endogenous control. ( D ) Western blot analysis with cellular lysates from HTR cells ( n = 3). GAPDH served as the loading control. ( E ) Organoids (ORGs) cultured under normoxia (norm; n = 5) or hypoxia (hypox; n = 4). Relative gene levels of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57), and TP53 (p53) are shown. The results are presented as relative quantification (RQ) with minimum and maximum range. GAPDH was used as endogenous control. ( F ) BeWo cells. Relative gene levels of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57), and TP53 (p53) are shown. The results ( n = 3) are presented as the relative quantification (RQ) with minimum and maximum range. GAPDH was used as the endogenous control. ( G ) Primary trophoblasts (pCTB) were isolated from healthy and preeclamptic (PE) donors. pCTBs from healthy donors were cultured under normoxia (norm; dot) or hypoxia (hypox, square), PE (triangle) pCTBs were grown under normoxia. The gene level of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57), and TP53 (p53) was evaluated ( n = 5). GAPDH was used as the housekeeping gene control. Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant; MV, mean value.

Journal: Cells

Article Title: Functional Analysis of p21 Cip1/ CDKN1A and Its Family Members in Trophoblastic Cells of the Placenta and Its Roles in Preeclampsia

doi: 10.3390/cells10092214

Figure Lengend Snippet: p21 expression is decreased in trophoblastic cell lines and in isolated primary trophoblasts under hypoxic conditions. Cells were grown under normoxia (norm, 21.4% O 2 ) or hypoxia (hypox, 1% O 2 ) for 48 h prior to RNA extraction or western blot analysis. ( A ) SGHPL-4 cells. Gene analysis of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57) and TP53 (p53) is shown. The results ( n = 3) are presented as relative quantification (RQ) with minimum and maximum range. GAPDH was used as the endogenous control. ( B ) Western blot analysis with cellular lysates from SGHPL-4 cells ( n = 3). GAPDH served as the loading control. ( C ) HTR cells. Gene analysis of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57), and TP53 (p53) is shown. The results ( n = 3) are presented as relative quantification (RQ) with minimum and maximum range. GAPDH was used as the endogenous control. ( D ) Western blot analysis with cellular lysates from HTR cells ( n = 3). GAPDH served as the loading control. ( E ) Organoids (ORGs) cultured under normoxia (norm; n = 5) or hypoxia (hypox; n = 4). Relative gene levels of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57), and TP53 (p53) are shown. The results are presented as relative quantification (RQ) with minimum and maximum range. GAPDH was used as endogenous control. ( F ) BeWo cells. Relative gene levels of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57), and TP53 (p53) are shown. The results ( n = 3) are presented as the relative quantification (RQ) with minimum and maximum range. GAPDH was used as the endogenous control. ( G ) Primary trophoblasts (pCTB) were isolated from healthy and preeclamptic (PE) donors. pCTBs from healthy donors were cultured under normoxia (norm; dot) or hypoxia (hypox, square), PE (triangle) pCTBs were grown under normoxia. The gene level of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57), and TP53 (p53) was evaluated ( n = 5). GAPDH was used as the housekeeping gene control. Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant; MV, mean value.

Techniques: Expressing, Isolation, RNA Extraction, Western Blot, Quantitative Proteomics, Control, Cell Culture

R316W-p57 mutation: effect on cell proliferation and cell cycle phase distribution. ( A ) Schematic representation of the structural domains of human p57 reporting the BWS-associated R316W mutation under analysis. ( B ) WB analysis of p57 in protein extracts of HEK293 cells transfected in triplicates with the empty vector (indicated as CTRL) and the plasmids encoding human WT- and R316W-p57 for 24 h and 48 h. Actin content was used to verify loading of equal amounts of proteins. ( C ) The graphs show the effect of the exogenous WT- and R316W-p57 with respect to CTRL on cell proliferation rate (upper panel) and on cell cycle phase distribution of HEK293 cells (bottom panel). The data shown are the mean of three independent experiments. The data were analyzed as indicated in Materials and Methods Section. The standard deviation (T bar) and the statistical significance ( p value) are reported.

Journal: International Journal of Molecular Sciences

Article Title: A Beckwith–Wiedemann-Associated CDKN1C Mutation Allows the Identification of a Novel Nuclear Localization Signal in Human p57 Kip2

doi: 10.3390/ijms22147428

Figure Lengend Snippet: R316W-p57 mutation: effect on cell proliferation and cell cycle phase distribution. ( A ) Schematic representation of the structural domains of human p57 reporting the BWS-associated R316W mutation under analysis. ( B ) WB analysis of p57 in protein extracts of HEK293 cells transfected in triplicates with the empty vector (indicated as CTRL) and the plasmids encoding human WT- and R316W-p57 for 24 h and 48 h. Actin content was used to verify loading of equal amounts of proteins. ( C ) The graphs show the effect of the exogenous WT- and R316W-p57 with respect to CTRL on cell proliferation rate (upper panel) and on cell cycle phase distribution of HEK293 cells (bottom panel). The data shown are the mean of three independent experiments. The data were analyzed as indicated in Materials and Methods Section. The standard deviation (T bar) and the statistical significance ( p value) are reported.

Article Snippet: The following primary antibodies were employed: anti-p57 (mouse monoclonal), anti-p57 (rabbit polyclonal), anti-LAMIN A/C (mouse monoclonal), anti-LDH (mouse monoclonal), and anti-β-actin (mouse monoclonal), all purchased from Santa Cruz Biotechnology, Inc., Heidelberg, Germany.

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Standard Deviation

$Subcellular localization of human WT- and R316W-p57. ( A ) WB analysis of the cytosolic and nuclear content of human p57 in HEK293 and HepG2 cell lines after 24 h of transfection with the empty vector (CTRL) and the plasmids encoding WT- and R316W-p57. Lamin A/C and LDH were analyzed to confirm the proper separation of nuclear and cytosolic fractions and as control for equal protein loading. ( B ) The subcellular localization of the WT and the mutated protein was investigated using immunofluorescence. HepG2 cells were stained with Alexa Fluor 488 (anti-p57 Ab, green), Hoechst 33342 (DNA, blue), and phalloidin (F-ACTIN, red). Representative images of cells overexpressing WT- and R316W-p57 were acquired using confocal microscopy with a 63x objective and are shown in panel ( B ). Scale bar 20 µm. For further details refer to the Materials and Methods Section.$

Journal: International Journal of Molecular Sciences

Article Title: A Beckwith–Wiedemann-Associated CDKN1C Mutation Allows the Identification of a Novel Nuclear Localization Signal in Human p57 Kip2

doi: 10.3390/ijms22147428

Figure Lengend Snippet: Subcellular localization of human WT- and R316W-p57. ( A ) WB analysis of the cytosolic and nuclear content of human p57 in HEK293 and HepG2 cell lines after 24 h of transfection with the empty vector (CTRL) and the plasmids encoding WT- and R316W-p57. Lamin A/C and LDH were analyzed to confirm the proper separation of nuclear and cytosolic fractions and as control for equal protein loading. ( B ) The subcellular localization of the WT and the mutated protein was investigated using immunofluorescence. HepG2 cells were stained with Alexa Fluor 488 (anti-p57 Ab, green), Hoechst 33342 (DNA, blue), and phalloidin (F-ACTIN, red). Representative images of cells overexpressing WT- and R316W-p57 were acquired using confocal microscopy with a 63x objective and are shown in panel ( B ). Scale bar 20 µm. For further details refer to the Materials and Methods Section.

Techniques: Transfection, Plasmid Preparation, Control, Immunofluorescence, Staining, Confocal Microscopy

$Nuclear localization of p57 is dependent on the C-terminal domain. ( A ) Schematic representation of the FL WT-p57 and the two N-terminal [(1–219)p57] and C-terminal [(121–316)p57] constructs. ( B ) The cellular localization of the exogenously expressed protein fragments was investigated using immunofluorescence as described in B. Representative immunofluorescence images of cells transfected with the plasmids encoding the N-terminal and C-terminal p57 fragments were reported. p57 (green), DNA (blue), and F-ACTIN (red). Scale bar 20 µm. For further details, refer to the Materials and Methods Section. ( C ) WB analysis of the cytosolic and nuclear content of p57 in HepG2 cells after 24h of transfection with the plasmids encoding FL WT-p57 (p57) and the two fragments (1–219)p57 and (121–316)p57. Lamin A/C and LDH were analyzed to confirm the proper separation of nuclear and cytosolic fractions and as control for equal protein loading.$

Journal: International Journal of Molecular Sciences

Article Title: A Beckwith–Wiedemann-Associated CDKN1C Mutation Allows the Identification of a Novel Nuclear Localization Signal in Human p57 Kip2

doi: 10.3390/ijms22147428

Figure Lengend Snippet: Nuclear localization of p57 is dependent on the C-terminal domain. ( A ) Schematic representation of the FL WT-p57 and the two N-terminal [(1–219)p57] and C-terminal [(121–316)p57] constructs. ( B ) The cellular localization of the exogenously expressed protein fragments was investigated using immunofluorescence as described in B. Representative immunofluorescence images of cells transfected with the plasmids encoding the N-terminal and C-terminal p57 fragments were reported. p57 (green), DNA (blue), and F-ACTIN (red). Scale bar 20 µm. For further details, refer to the Materials and Methods Section. ( C ) WB analysis of the cytosolic and nuclear content of p57 in HepG2 cells after 24h of transfection with the plasmids encoding FL WT-p57 (p57) and the two fragments (1–219)p57 and (121–316)p57. Lamin A/C and LDH were analyzed to confirm the proper separation of nuclear and cytosolic fractions and as control for equal protein loading.

Techniques: Construct, Immunofluorescence, Transfection, Control

$Cellular distribution of the (121-316)p57, (121-316)R316Wp57, and the truncated fragment (121–311)p57. ( A ) Schematic representation of the human C-term (121–316)p57 fragment, the mutagenized (121–316)R316Wp57, and the truncated (121–311)p57 protein fragments. ( B ) Representative immunofluorescence images of HepG2 cells transfected with the plasmids encoding the indicated proteins listed in panel A. p57 (green), DNA (blue), and F-ACTIN (red). Scale bar 20 µm. For further details refer to the Materials and Methods Section. ( C ) WB analysis of the cytosolic and nuclear content of p57 in HEK293 after 24h of transfection with the plasmids represented in panel A. Lamin A/C and LDH were analyzed to confirm the proper separation of nuclear and cytosolic fractions and as control for equal protein loading.$

Journal: International Journal of Molecular Sciences

Article Title: A Beckwith–Wiedemann-Associated CDKN1C Mutation Allows the Identification of a Novel Nuclear Localization Signal in Human p57 Kip2

doi: 10.3390/ijms22147428

Figure Lengend Snippet: Cellular distribution of the (121-316)p57, (121-316)R316Wp57, and the truncated fragment (121–311)p57. ( A ) Schematic representation of the human C-term (121–316)p57 fragment, the mutagenized (121–316)R316Wp57, and the truncated (121–311)p57 protein fragments. ( B ) Representative immunofluorescence images of HepG2 cells transfected with the plasmids encoding the indicated proteins listed in panel A. p57 (green), DNA (blue), and F-ACTIN (red). Scale bar 20 µm. For further details refer to the Materials and Methods Section. ( C ) WB analysis of the cytosolic and nuclear content of p57 in HEK293 after 24h of transfection with the plasmids represented in panel A. Lamin A/C and LDH were analyzed to confirm the proper separation of nuclear and cytosolic fractions and as control for equal protein loading.

Techniques: Immunofluorescence, Transfection, Control

$Relevance of the last five p57 residues in the nuclear localization of the protein. ( A ) Schematic representation of FL WT-p57 and (1–311)-p57. ( B ) WB analysis of p57 cytoplasmic/nuclear distribution in HEPG2 cells transfected for 24 h with the empty vector (CTRL) and the plasmids encoding the WT- and (1–311)-p57. Lamin A/C and LDH were employed to confirm the proper separation of nuclear and cytosolic fractions and as control for equal protein loading. ( C ) Representative immunofluorescence image of the overexpressed (1–311)p57 in HepG2 cells. p57 (green), DNA (blue), and F-ACTIN (red). Scale bar 20 µm. For further details refer to the Materials and Methods Section.$

Journal: International Journal of Molecular Sciences

Article Title: A Beckwith–Wiedemann-Associated CDKN1C Mutation Allows the Identification of a Novel Nuclear Localization Signal in Human p57 Kip2

doi: 10.3390/ijms22147428

Figure Lengend Snippet: Relevance of the last five p57 residues in the nuclear localization of the protein. ( A ) Schematic representation of FL WT-p57 and (1–311)-p57. ( B ) WB analysis of p57 cytoplasmic/nuclear distribution in HEPG2 cells transfected for 24 h with the empty vector (CTRL) and the plasmids encoding the WT- and (1–311)-p57. Lamin A/C and LDH were employed to confirm the proper separation of nuclear and cytosolic fractions and as control for equal protein loading. ( C ) Representative immunofluorescence image of the overexpressed (1–311)p57 in HepG2 cells. p57 (green), DNA (blue), and F-ACTIN (red). Scale bar 20 µm. For further details refer to the Materials and Methods Section.

Techniques: Transfection, Plasmid Preparation, Control, Immunofluorescence

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

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

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

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

Techniques: Immunofluorescence, Double Immunostaining

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