anti ctmp antibody  (Cell Signaling Technology Inc)

Journal: Journal of Cellular Physiology

Article Title: Carboxyl Terminal Modulator Protein Induces Cell Senescence and Is Upregulated With Aging by Zic2 in Rats

doi: 10.1002/jcp.70007

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Article Snippet: Rabbit polyclonal anti‐CTMP , Cell Signaling Technology , 4612s.

Techniques: Plasmid Preparation, Luciferase, Transfection, Staining, Protease Inhibitor

Journal: Heliyon

Article Title: PRAME promotes proliferation of multiple myeloma cells through CTMP/Akt/p21/CCND3 axis by ubiquitinating CTMP and p21

doi: 10.1016/j.heliyon.2024.e34094

Figure Lengend Snippet: CTMP and p21 were novel PRAME-interacting proteins in MM cells. (A) Pathway enrichment results based on Metascape analysis of top 100 proteins with reduced expression followed by PRAME knockdown. Top 20 pathways were listed. Regulation of mitotic cell cycle was most significantly enriched. (B) Proteins interacting with cyclins analyzed by STRING database. In addition to proteins of cytokinin-dependent kinase (CDK) family, cytokinin-dependent kinase inhibitors (such as CDKN1A and CDKN1B ) are attractive. (C) CCND3 and p21 expression levels tested by Western blot in MM cell lines compared PRAME knockdown or overexpression groups to control groups (related to - ). LP-1 was not applied for subsequent protein detection experiments for the low protein expression level of p21 (related to - ). (D) Flow diagram of IP-MS of RPMI8226 cells. (E) Western blot verified the CTMP/Akt/p21/CCND3 pathway in MM cell lines RPMI8226, KM-3 and SKO-007 (related to - ). Protein expression levels of CTMP and p21 were increased, and p -Akt and CCND3 were decreased followed by PRAME knockdown. (F) PRAME interacted with CTMP, p21 and Cul2 at the endogenous level. Cells were harvested and subjected to immunoprecipitation with anti-PRAME Ab and immunoblotting with anti-CTMP, anti-p21 and anti-Cul2 Ab (related to - ). (G) Higher PRAME transcript levels correlated with lower THEM4 and CDKN1A transcript levels in MM patients' plasma cells.

Article Snippet: Cell extracts were incubated with primary antibodies PRAME (ab219650, Abcam) for co-immunoprecipitation (co-IP), p21 (ab109520, Abcam) and CTMP (14692-1-AP, Proteintech, Hubei, China) for endogenous ubiquitination assay or control IgG in a rotating incubator overnight at 4 °C, followed by incubation with protein A/G Plus-Agarose (sc2003, Santa Cruz Biotechnology, TX, USA) for another 2 h. The immunoprecipitates were washed three times with lysis buffer and analyzed by immunoblotting with anti-CTMP and anti-p21 for co-IP assay, as well as anti-ubiquitin (sc8017, Santa Cruz Biotechnology, TX, USA) for ubiquitination testing.

Techniques: Expressing, Knockdown, Western Blot, Over Expression, Control, Protein-Protein interactions, Immunoprecipitation, Clinical Proteomics

Journal: Heliyon

Article Title: PRAME promotes proliferation of multiple myeloma cells through CTMP/Akt/p21/CCND3 axis by ubiquitinating CTMP and p21

doi: 10.1016/j.heliyon.2024.e34094

Figure Lengend Snippet: PRAME degrades CTMP and p21 through UPS. (A, B) CTMP and p21 were degraded by proteasomes. RPMI8226 cells were treated with 10 μmol/L MG-132 versus DMSO in combination with 50 μg/mL CHX for indicated time (0, 2h, 4h and 8h, respectively), and then were subjected to immunoblotting using anti-CTMP and anti-p21 with GAPDH as a loading control (related to - ). (C, D) The expression of CTMP and p21 in A and B was quantified by densitometric analysis using ImageJ software, respectively. Data are the means ± SD of 3 assays. ns , no significance; * P < 0.05; ** P < 0.01; *** P < 0.001. (E, F) PRAME mediated polyubiquitination of CTMP and p21 in RPMI8226 cells. PRAME knockdown and control cell models were used. Cells were extracted and subjected to immunoprecipitation with anti-CTMP and anti-p21 Ab and immunoblotting with anti-ubiquitin Ab with MG-132 for 2h and 6h, respectively (related to - ).

Article Snippet: Cell extracts were incubated with primary antibodies PRAME (ab219650, Abcam) for co-immunoprecipitation (co-IP), p21 (ab109520, Abcam) and CTMP (14692-1-AP, Proteintech, Hubei, China) for endogenous ubiquitination assay or control IgG in a rotating incubator overnight at 4 °C, followed by incubation with protein A/G Plus-Agarose (sc2003, Santa Cruz Biotechnology, TX, USA) for another 2 h. The immunoprecipitates were washed three times with lysis buffer and analyzed by immunoblotting with anti-CTMP and anti-p21 for co-IP assay, as well as anti-ubiquitin (sc8017, Santa Cruz Biotechnology, TX, USA) for ubiquitination testing.

Techniques: Western Blot, Control, Expressing, Software, Knockdown, Immunoprecipitation, Ubiquitin Proteomics

Journal: Heliyon

Article Title: PRAME promotes proliferation of multiple myeloma cells through CTMP/Akt/p21/CCND3 axis by ubiquitinating CTMP and p21

doi: 10.1016/j.heliyon.2024.e34094

Figure Lengend Snippet: PRAME increases sensitivity of MM cells to bortezomib by targeting CTMP and p21. (A) Apoptosis analysis by flow cytometry in PRAME knockdown and overexpression cells with corresponding vector controls after bortezomib treating for 48h (LP-1, 10 nM; RPMI8226 20 nM). Data are the means ± SD of 3 assays. ns , no significance; * P < 0.05; ** P < 0.01; *** P < 0.001. (B) CTMP and p21 were degraded more intensely by proteasomes in PRAME high expression group than that in PRAME low expression group. PRAME knockdown and the control RPMI8226 cells were treated with 10 μmol/L MG-132 versus DMSO in combination with 50 μg/mL CHX for indicated time (0, 2h, 4h and 8h, respectively), and then were subjected to immunoblotting using anti-PRAME, anti-CTMP and anti-p21 with GAPDH as a loading control (related to - ). (C, D) The expression of CTMP and p21 in B was quantified by densitometric analysis using ImageJ software, respectively. Significance was shown between the fold changes of shCtrl PRAME MG132+CHX/DMSO + CHX group compared to shPRAME MG132+CHX/DMSO + CHX group at each time point. Data are the means ± SD of 3 assays. ns , no significance; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Cell extracts were incubated with primary antibodies PRAME (ab219650, Abcam) for co-immunoprecipitation (co-IP), p21 (ab109520, Abcam) and CTMP (14692-1-AP, Proteintech, Hubei, China) for endogenous ubiquitination assay or control IgG in a rotating incubator overnight at 4 °C, followed by incubation with protein A/G Plus-Agarose (sc2003, Santa Cruz Biotechnology, TX, USA) for another 2 h. The immunoprecipitates were washed three times with lysis buffer and analyzed by immunoblotting with anti-CTMP and anti-p21 for co-IP assay, as well as anti-ubiquitin (sc8017, Santa Cruz Biotechnology, TX, USA) for ubiquitination testing.

Techniques: Flow Cytometry, Knockdown, Over Expression, Plasmid Preparation, Expressing, Control, Western Blot, Software

Journal: Heliyon

Article Title: PRAME promotes proliferation of multiple myeloma cells through CTMP/Akt/p21/CCND3 axis by ubiquitinating CTMP and p21

doi: 10.1016/j.heliyon.2024.e34094

Figure Lengend Snippet: Mechanism diagram exhibits the role of PRAME in promoting cell proliferation and increasing sensitivity to bortezomib in MM cells. On the CTMP/Akt/p21/CCND3 signal pathway, PRAME plays the role of Cul2-dependent substrate recognizing receptor protein, and interacts with its target CTMP, labeling it with ubiquitin, and making it chemotactic to proteasome and degraded through UPS. The degradation of CTMP contributes to phosphorylation and activation of Akt, which leads to blocking of p21 expression. At the same time, PRAME directly takes p21 as its ubiquitination substrate, directing it degraded through the UPS. Both of the above-mentioned two pathways decrease p21 levels, thus leading to accumulation of CCND3 and promoting the cell cycle progression and cell proliferation.

Article Snippet: Cell extracts were incubated with primary antibodies PRAME (ab219650, Abcam) for co-immunoprecipitation (co-IP), p21 (ab109520, Abcam) and CTMP (14692-1-AP, Proteintech, Hubei, China) for endogenous ubiquitination assay or control IgG in a rotating incubator overnight at 4 °C, followed by incubation with protein A/G Plus-Agarose (sc2003, Santa Cruz Biotechnology, TX, USA) for another 2 h. The immunoprecipitates were washed three times with lysis buffer and analyzed by immunoblotting with anti-CTMP and anti-p21 for co-IP assay, as well as anti-ubiquitin (sc8017, Santa Cruz Biotechnology, TX, USA) for ubiquitination testing.

Techniques: Labeling, Ubiquitin Proteomics, Phospho-proteomics, Activation Assay, Blocking Assay, Expressing

Journal: Nitric oxide : biology and chemistry

Article Title: Simvastatin restores pulmonary endothelial function in the setting of pulmonary over-circulation

doi: 10.1016/j.niox.2023.11.007

Figure Lengend Snippet: PAECs were transduced with increasing multiplicity of infection (MOI) from 1 to 20 viral particles/cell exposure of an adenovirus expressing CTMP and Western blot analysis was used to determine the increase in CTMP protein levels (A). An MOI of 10 was then used to transduce shunt PAECs for 48 h. CTMP over-expression increases NO generation in shunt PAECs by both DAF fluorescence intensity and bioavailable NOx in cell media (B, C). The increase in NO generation correlates with a decrease in the levels of eNOS localized to the mitochondria as determined by colocalization of eNOS and mitochondria comparing mean Pearson coefficients derived from fluorescence microscopy (D) and reduced NOS-derived superoxide from PAECs (E). Values are mean ± SEM. N = 6 *P < 0.05 by two tailed t -test comparing AdCTMP to vehicle treated controls.

Article Snippet: Membranes were then probed at room temperature with primary antibodies to Akt1 (Cell Signaling, Danvers, MA), pS473 Akt1 (Cell Signaling, Danvers, MA), eNOS (BD Transduction, Franklin Lakes, NJ), pS1177 eNOS (BD Transduction, Franklin Lakes, NJ), pS617 eNOS (Millipore, Burlington, MA and Invitrogen, Waltham, MA), and CTMP (Cell Signaling, Danvers, MA).

Techniques: Transduction, Infection, Expressing, Western Blot, Over Expression, Fluorescence, Derivative Assay, Microscopy, Two Tailed Test