Journal: International Journal of Molecular Sciences

Article Title: Lipopolysaccharide-Activated Macrophages Suppress Cellular Senescence and Promote Rejuvenation in Human Dermal Fibroblasts

doi: 10.3390/ijms26157061

Figure Lengend Snippet: Expression of the p16 and p21 genes in Young cells. The schematic protocol (1)–(7) is shown at the top of the figure. The test conditions in figures ( A – D ) correspond to the schematic protocol (1)–(7). Young cells were cultured for 24 h after replacing half of the medium with EMEM, Old cell-conditioned medium (Old CM), or THP/LPS-conditioned medium (THP/LPS-CM; 0–1000 ng/mL). ( A , B ) Total RNA was extracted, and the relative mRNA expression levels of p16 ( A ) and p21 ( B ) were measured by Quantitative real-time PCR (RT-qPCR) using GAPDH as a housekeeping gene. ( C , D ) Young cells were treated with EMEM or THP/LPS-CM (0, 10, or 100 ng/mL) for 24 h. Cells were lysed, and 12.5 μg of total protein per lane was separated by SDS-PAGE, followed by western blot analysis using antibodies against P16 ( C ) or P21 ( D ). Representative western blot images are shown in the boxed regions of panels C and D. Band intensities were normalized to β-actin and quantified. Data are presented as mean ± SD (n = 3). Different letters (a–c) indicate statistically significant differences ( p < 0.05, ANOVA, Tukey–Kramer).

Article Snippet: Proteins were resolved via 12.5% SDS-PAGE at 30 mA for 90 min and transferred onto PVDF membranes at 300 mA for 1 h. Membranes were then blocked with PBS containing 0.1% Tween-20 and 5% skim milk for 1 h, followed by incubation overnight at 4 °C with primary antibodies against P21 Waf1/Cip1 (1:1000) and β-actin (1:2000; both from Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, SDS Page, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Lipopolysaccharide-Activated Macrophages Suppress Cellular Senescence and Promote Rejuvenation in Human Dermal Fibroblasts

doi: 10.3390/ijms26157061

Figure Lengend Snippet: Changes in P16, P21, and Ki-67 expression in senescent NB1RGB cells treated with THP/LPS-CM. The schematic protocol (1)–(7) is shown at the top of the figure. The test conditions in figures ( A – D ) correspond to the schematic protocol (1)–(7). ( A ) Young and Old NB1RGB cells were treated with EMEM, THP/LPS (0 ng/mL)-CM, or THP/LPS (100 ng/mL)-CM for 24 h. Relative mRNA expression levels of p16 , p21 , and Ki-67 were analyzed by RT-qPCR and normalized to the levels in Young cells. ( B ) Old cells were treated with LPS (10, 100, or 1000 ng/mL) for 24 h, and the expression levels of p16 , p21 , and Ki-67 were assessed by RT-qPCR. In both ( A , B ), bars represent mean ± SD (n = 5). Different letters (a–c) indicate statistically significant differences ( p < 0.05). ( C ) Young and Old cells were treated with EMEM, THP/LPS (0 ng/mL)-CM, or THP/LPS (100 ng/mL)-CM for 24 h, then immunofluorescence staining was performed using antibodies against P21 and Ki-67 (n = 5). Representative fluorescence images were acquired using a 20× objective lens with 2× digital zoom. In P21 and Ki-67 single-staining panels green = P21 or Ki-67, red = actin, and blue = nuclei. In double-staining panels green = P21, red = Ki-67, and blue = nuclei. Scale bars = 50 μm. ( D ) Based on the immunofluorescence images, the percentages of P21-positive cells, Ki-67-positive cells, and total cell counts per field were quantified. Data are expressed as mean ± SD (n = 5). Different letters (a–c) indicate statistically significant differences ( p < 0.05, ANOVA, Tukey–Kramer).

Article Snippet: Proteins were resolved via 12.5% SDS-PAGE at 30 mA for 90 min and transferred onto PVDF membranes at 300 mA for 1 h. Membranes were then blocked with PBS containing 0.1% Tween-20 and 5% skim milk for 1 h, followed by incubation overnight at 4 °C with primary antibodies against P21 Waf1/Cip1 (1:1000) and β-actin (1:2000; both from Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Fluorescence, Double Staining

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: FOXO4-DRI regulates endothelial cell senescence via the P53 signaling pathway

doi: 10.3389/fbioe.2025.1729166

Figure Lengend Snippet: FOXO4-DRI can effectively maintain vascular function in naturally aging mice. (A) Western blot experiments were conducted and quantitatively analyzed to determine the changes in protein levels of Ki-67, Lamin B, P21, P16, and γ-H2AX in the aortas of naturally aged mice after FOXO4-DRI or PBS treatment. n ≥ 4 per group. (B) RT-qPCR analysis was performed to examine the mRNA levels of Il-1β, Il-6, Cxcl15 , and Tnf-α in the aortas of naturally aged mice after FOXO4-DRI and PBS treatments. n = 3 per group. (C) Representative SA-β-Gal staining and quantitative analysis were performed to determine the number of positive cells in the aortas of naturally aged mice after FOXO4-DRI and PBS treatments. n = 5 per group. Scale bar = 100 μm. (D) Representative HE staining and quantitative analysis were performed to evaluate the thickness of the aortas in naturally aged mice after FOXO4-DRI and PBS treatments. n = 5 per group. Scale bar = 100 μm. (E) Color Doppler imaging and analysis were performed to detect and analyze the structural and blood flow conditions of the aortas in naturally aged mice after FOXO4-DRI and PBS treatments. n = 6 per group. (F) Representative DHE staining and quantitative analysis were performed to assess the ROS levels in the aortas of naturally aged mice after FOXO4-DRI and PBS treatments. n = 5 per group. Scale bar = 100 μm. Data are presented as mean ± SEM and analyzed using a two-tailed Student’s t-test. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

Article Snippet: The primary antibodies employed were: P53 (Proteintech, 60283-2-Ig), pSer46-P53 (CST, 2521), FOXO4 (Proteintech, 21535-1-AP), P21 (CST, 2947), P16 (CST, 18769), BAX (CST, 2772), BCL2 (CST, 3498), Ki-67 (Proteintech, 84192-4-RR), Lamin B (CST, 13435), Cleaved Caspase 3 (CST, 9664), γ-H2AX (Abcam, ab81299), and GAPDH (Abcam, ab8245) as an internal reference for normalizing protein expression levels.

Techniques: Western Blot, Quantitative RT-PCR, Staining, Imaging, Two Tailed Test

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: FOXO4-DRI regulates endothelial cell senescence via the P53 signaling pathway

doi: 10.3389/fbioe.2025.1729166

Figure Lengend Snippet: FOXO4-DRI can effectively maintain vascular function in progeroid mice. (A) Western blot experiments were conducted and quantitatively analyzed to determine the changes in protein levels of Ki-67, Lamin B, P21, P16, and γ-H2AX in the aortas of progeroid mice after FOXO4-DRI or PBS treatment. n ≥ 4 per group. (B) RT-qPCR analysis was performed to examine the mRNA levels of Il-1β , Il-6 , Cxcl15 , and Tnf-α in the aortas of progeroid mice after FOXO4-DRI and PBS treatments. n = 3 per group. (C) Representative SA-β-Gal staining and quantitative analysis were performed to determine the number of positive cells in the aortas of progeroid mice after FOXO4-DRI and PBS treatments. Scale bar = 100 μm. n = 5 per group. (D) Representative HE staining and quantitative analysis were performed to evaluate the thickness in the aortas of progeroid mice after FOXO4-DRI and PBS treatments. Scale bar = 100 μm. n = 5 per group. (E) Color Doppler imaging and analysis were performed to detect and analyze the structural and blood flow conditions in the aortas of progeroid mice after FOXO4-DRI and PBS treatments. n = 5 per group. (F) Representative DHE staining and quantitative analysis were performed to assess the ROS levels in the aortas of progeroid mice after FOXO4-DRI and PBS treatments. n = 5 per group. Scale bar = 100 μm. Data are presented as mean ± SEM and analyzed using a two-tailed Student’s t-test. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

Article Snippet: The primary antibodies employed were: P53 (Proteintech, 60283-2-Ig), pSer46-P53 (CST, 2521), FOXO4 (Proteintech, 21535-1-AP), P21 (CST, 2947), P16 (CST, 18769), BAX (CST, 2772), BCL2 (CST, 3498), Ki-67 (Proteintech, 84192-4-RR), Lamin B (CST, 13435), Cleaved Caspase 3 (CST, 9664), γ-H2AX (Abcam, ab81299), and GAPDH (Abcam, ab8245) as an internal reference for normalizing protein expression levels.

Techniques: Western Blot, Quantitative RT-PCR, Staining, Imaging, Two Tailed Test

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: FOXO4-DRI regulates endothelial cell senescence via the P53 signaling pathway

doi: 10.3389/fbioe.2025.1729166

Figure Lengend Snippet: Glucose and oxygen deprivation can induce endothelial cell senescence. (A) Western blot analysis was performed to quantify the changes in protein levels of Ki-67, Lamin B, FOXO4, P53, P21, P16, and γ-H2AX in HUVECs following OGD treatment, with n ≥ 4 per group. (B) Representative immunofluorescence staining was conducted to analyze the content and localization changes of Ki-67, P21, γ-H2AX, and P53 different groups of HUVECs, with n = 5 per group. (C) Subcellular localization of FOXO4 in different groups of HUVECs was analyzed through representative immunofluorescence staining (FOXO4 in red, DAPI in blue), with n = 5 per group, scale bar = 40 μm. (D) Representative SA-β-GAL staining and quantification were performed to evaluate the number of positive cells in HUVECs after OGD treatment, with n = 5 per group, scale bar = 100 μm. (E) Tube formation assays analyzed relative tube lengths in different groups following OGD treatment, with n = 5 per group, scale bar = 400 μm. (F) Scratch assays were conducted to assess endothelial cell migration coverage at 0, 12, 24, and 36 h across different groups, with n = 5 per group, scale bar = 400 μm. Data are represented as mean ± SEM. Statistical analysis assessed by 2-way RM ANOVA (G) Representative DHE staining and quantification evaluated the number of DHE-positive cells in different groups, with n = 5 per group, scale bar = 100 μm. (H) RT-qPCR analysis was performed in HUVECs following OGD treatment to measure the mRNA levels of IL-6 , IL-8 , IL-1β , and TNF-α , with n = 3 per group. Data are presented as mean ± SEM and analyzed using a two-tailed Student’s t-test. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

Article Snippet: The primary antibodies employed were: P53 (Proteintech, 60283-2-Ig), pSer46-P53 (CST, 2521), FOXO4 (Proteintech, 21535-1-AP), P21 (CST, 2947), P16 (CST, 18769), BAX (CST, 2772), BCL2 (CST, 3498), Ki-67 (Proteintech, 84192-4-RR), Lamin B (CST, 13435), Cleaved Caspase 3 (CST, 9664), γ-H2AX (Abcam, ab81299), and GAPDH (Abcam, ab8245) as an internal reference for normalizing protein expression levels.

Techniques: Western Blot, Immunofluorescence, Staining, Migration, Quantitative RT-PCR, Two Tailed Test

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: FOXO4-DRI regulates endothelial cell senescence via the P53 signaling pathway

doi: 10.3389/fbioe.2025.1729166

Figure Lengend Snippet: FOXO4-DRI can alleviate endothelial cell senescence induced by OGD. (A) Western blot analysis was conducted to quantify the changes in protein levels of Ki-67, Lamin B, P21, P16, and γ-H2AX in senescent HUVECs following treatment with FOXO4-DRI or PBS, with n ≥ 4 per group. (B) Representative immunofluorescence analysis (green for Ki-67, blue for DAPI) was performed to assess the number of Ki-67 positive HUVECs in different groups, with n = 5 per group and scale bar = 40 μm. (C) Fluorescent staining was carried out to analyze the content and localization changes of P21 and γ-H2AX in different groups, with n = 5 per group and scale bar = 40 μm. (D) Representative SA-β-GAL staining and quantification were performed to evaluate the number of positive cells in different groups, with n = 5 per group and scale bar = 100 μm. (E) Tube formation assays were conducted to analyze relative tube lengths in different groups, with n = 5 per group and scale bar = 400 μm. (F) Representative DHE staining and quantification were carried out to evaluate the number of DHE-positive cells in different groups, with n = 5 per group and scale bar = 100 μm. (G) Scratch assays were performed to assess endothelial cell migration coverage at 0, 12, 24, and 36 h in different groups, with n = 5 per group and scale bar = 400 μm. Data are represented as mean ± SEM. Statistical analysis assessed by 2-way RM ANOVA. (H) RT-qPCR analysis was performed to detect mRNA levels of IL-6 , IL-8 , IL-1β , and TNF-α in different groups, with n = 3 per group. Data are presented as mean ± SEM and analyzed using a two-tailed Student’s t-test. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

Article Snippet: The primary antibodies employed were: P53 (Proteintech, 60283-2-Ig), pSer46-P53 (CST, 2521), FOXO4 (Proteintech, 21535-1-AP), P21 (CST, 2947), P16 (CST, 18769), BAX (CST, 2772), BCL2 (CST, 3498), Ki-67 (Proteintech, 84192-4-RR), Lamin B (CST, 13435), Cleaved Caspase 3 (CST, 9664), γ-H2AX (Abcam, ab81299), and GAPDH (Abcam, ab8245) as an internal reference for normalizing protein expression levels.

Techniques: Western Blot, Immunofluorescence, Staining, Migration, Quantitative RT-PCR, Two Tailed Test

Journal: Antioxidants

Article Title: Epicatechin Gallate Ameliorates UVB-Induced Photoaging by Inhibiting p38α-Mediated Autophagy and Oxidative Stress

doi: 10.3390/antiox15020180

Figure Lengend Snippet: Effects of ECG on UVB-induced senescence markers in HaCaT cells. ( A , B ) Cell cycle distribution analysis of HaCaT cells by flow cytometry. The histogram in ( A ) shows DNA content (propidium iodide staining intensity) on the x-axis and cell count on the y-axis. The stacked bar chart in ( B ) quantifies the percentage of cells in different cell cycle phases (G0/G1, S, and G2/M). ( C ) Relative mRNA expression levels of p53 , p16 , p21 , IL6 , and TNF-α determined by RT-qPCR. ( D , E ) Representative Western blots and densitometric analysis of p53, p16, and p21 proteins. ( F , I ) Representative immunofluorescence images of γH2AX and Lamin B1 (scale bar: 50 μm). γH2AX foci are shown in green, Lamin B1 is shown in red, and nuclei are counterstained with DAPI (blue). White arrows indicate nuclei with damage. ( G ) Representative images of SA-β-gal staining in HaCaT cells (scale bar: 200 μm). ( H ) Quantitative analysis of SA-β-gal positive cells. Data are presented as the mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: Rabbit anti-Beclin1 (BECN1) antibody (cat. #11306), mouse anti-tubulin antibody (cat. #66031-1-IG), rabbit anti-LaminB1 (LMNB1) antibody (cat. #12987-1-AP), rabbit anti-Cyclin-Dependent Kinase Inhibitor 1A (p21, CDKN1A) antibody (cat. #10355-1-AP) and rabbit anti-LC3 antibody (cat. #14600) were purchased from Proteintech group (Wuhan, China).

Techniques: Flow Cytometry, Staining, Cell Characterization, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence

Journal: bioRxiv

Article Title: A feed-forward loop between niche adenosine and Gzmk⁺ CD8 T cells propagates systemic inflammaging

doi: 10.64898/2026.03.18.712515

Figure Lengend Snippet: (A-D) The analysis of male 19 months WT or Gzmk -/- mice (male). (A) Staining of SA-β-gal in liver and hippocampus. (B) H&E staining in liver, lung and kidney. (C) SA-β-gal staining for WAT from indicated mice. (D) Western blot analysis of the expression of p16, p21 and p53 in liver. (E) The bioluminescence imaging of indicated mice (15 months, female) by injecting luciferase substrates. (F-G) CD8 T cells (4 x10 6 ) from old or old Gzmk -/- mice were adoptively transferred into young p16 Ink4a -luciferase reporter mice, one month later, the luciferase activity was measured (F), and the expression of the p16 -driven luciferase reporter in indicated tissues were measured by RT-qPCR.

Article Snippet: Primary antibodies used in this study: Mouse anti-CDKN2A/p16 (Santa Cruz Biotechnology, cat# sc-1661, 1:1000) Mouse anti-CDKN1A p21 (Santa Cruz Biotechnology, cat# sc-6246, 1:1000) Mouse anti-p53 (Santa Cruz Biotechnology, cat# sc-98, 1:1000) Mouse anti-GAPDH (Absin, abs830030,1:10000) Rabbit anti-γH2A.X (Cell Signaling Technology, cat# 9718S, 1:1000)

Techniques: Staining, Western Blot, Expressing, Imaging, Luciferase, Activity Assay, Quantitative RT-PCR

Journal: bioRxiv

Article Title: A feed-forward loop between niche adenosine and Gzmk⁺ CD8 T cells propagates systemic inflammaging

doi: 10.64898/2026.03.18.712515

Figure Lengend Snippet: Old mice (19 months) were administrated with SCH or PPACK for one-month. The level of IL-6 and TNFα (A), ALT and AST (B) in plasma from indicated mice were determined by ELSIA. (C) Staining of SA-β-gal in liver. (D) H&E staining of lungs from indicated mice. (E) Staining of SA-β-gal in hippocampus from indicated mice. (F-G) Western blot analysis of P16, P21 and P53 at liver and brain from indicated mice.

Article Snippet: Primary antibodies used in this study: Mouse anti-CDKN2A/p16 (Santa Cruz Biotechnology, cat# sc-1661, 1:1000) Mouse anti-CDKN1A p21 (Santa Cruz Biotechnology, cat# sc-6246, 1:1000) Mouse anti-p53 (Santa Cruz Biotechnology, cat# sc-98, 1:1000) Mouse anti-GAPDH (Absin, abs830030,1:10000) Rabbit anti-γH2A.X (Cell Signaling Technology, cat# 9718S, 1:1000)

Techniques: Clinical Proteomics, Staining, Western Blot

Journal: Oncogene

Article Title: Myostatin inhibits rhabdomyosarcoma cell proliferation through an Rb-independent pathway.

doi: 10.1038/sj.onc.1207144

Figure Lengend Snippet: Figure 5 Myostatin does not alter the levels of the CKIs p21, p15, p16 or p27 in RMS cells. Western blots showing the levels of p21, p15, p16 and p27 protein in RD cells cultured with ( þ ) or without () myostatin for 24 and 48 h. p21, p15, p16 and p27 immunoreactive proteins were detected using their respective antibodies. Tubulin protein levels, detected by antitubulin anti- bodies, are included to show even loadings

Article Snippet: The following primary antibodies were used for immunoblotting; myostatin: 1 : 2000 dilution of rabbit polyclonal antimyostatin antibody (Thomas et al., 2000); p15, 1 : 400 dilution of purified rabbit polyclonal anti-p15 antibody (sc-613; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); p16, 1 : 400 dilution of purified rabbit polyclonal antip16 antibody (sc-1207; Santa Cruz Biotechnology Inc.); p21, 1 : 400 dilution of purified mouse monoclonal anti-p21 antibody (SX118; PharMingen, CA, USA); p27, 1 : 400 dilution of purified mouse monoclonal anti-p27 antibody (sc-1641; Santa Cruz Biotechnology Inc.); Cdk2, 1 : 400 dilution of purified mouse monoclonal anti-Cdk2 antibody (sc-2648; Santa Cruz Biotechnology Inc.); Cdk4, 1 : 400 dilution of purified rabbit polyclonal anti-Cdk4 antibody (sc-601; Santa Cruz Biotechnology Inc.); cyclin-D1, 1 : 400 dilution of purified mouse monoclonal anticyclin-D1 antibody (sc-8396; Santa Cruz Biotechnology Inc.); cyclin-E, 1 : 400 dilution of purified rabbit polyclonal anticyclin-E antibody (sc-481; Santa Cruz Biotechnology Inc.); a-tubulin, 1 : 3000 dilution of purified mouse monoclonal anti-a-tubulin antibody (DM 1A; Sigma); Rb, 1 : 200 dilution of purified mouse monoclonal anti-pRb antibody (G3-245; PharMingen); E2F1, 1 : 250 dilution of purified mouse monoclonal anti-E2F1 antibody (KH95; PharMingen); NPAT, 1 : 250 dilution of purified mouse monoclonal antiNPAT antibody (C27; Transduction Laboratories) or 1 : 2000 rabbit polyclonal anti-NPAT antibody (Zhao et al., 2000).

Techniques: Western Blot, Cell Culture

Journal: Nutrients

Article Title: The Effect of Resveratrol or Curcumin on Head and Neck Cancer Cells Sensitivity to the Cytotoxic Effects of Cisplatin

doi: 10.3390/nu12092596

Figure Lengend Snippet: The total p21 protein expression (pg/mL) in normal cell line-HUVEC and in tumor cell line-PE/CA-PJ49 cells treated 24 h with CisPt and/or RSV, CRM. The experiments were performed in triplicates. Results are expressed as mean values of three determinations ± standard deviation (SD). (** p < 0.005, *** p < 0.0005; **** p < 0.00005).

Article Snippet: DuoSetR IC ELISA-Human Total p21/CIP1/CDKN1A(: DYC1047-2) was purchased from R&D Systems Inc. and contains the basic components required for the development of sandwich ELISAs to measure human p21 protein also known as CIP1 and CDKN1A in cell lysates.

Techniques: Expressing, Standard Deviation

Journal: Nutrients

Article Title: The Effect of Resveratrol or Curcumin on Head and Neck Cancer Cells Sensitivity to the Cytotoxic Effects of Cisplatin

doi: 10.3390/nu12092596

Figure Lengend Snippet: The effects of CisPt, RSV, CRM treatment applied alone or in combination for 24 h on the level of p21 protein expression (n-fold p21protein expression) in normal cell line-Huvec and in tumor cell line-PE/CA-PJ49. The n-fold p21expression was calculated using the formula: n-fold p21expression = p21 pg/mL treatment/p21 pg/mL control.

Article Snippet: DuoSetR IC ELISA-Human Total p21/CIP1/CDKN1A(: DYC1047-2) was purchased from R&D Systems Inc. and contains the basic components required for the development of sandwich ELISAs to measure human p21 protein also known as CIP1 and CDKN1A in cell lysates.

Techniques: Expressing, Control

Journal: Nutrients

Article Title: The Effect of Resveratrol or Curcumin on Head and Neck Cancer Cells Sensitivity to the Cytotoxic Effects of Cisplatin

doi: 10.3390/nu12092596

Figure Lengend Snippet: The effect of treatment with CisPt, RSV, CRM applied independently or in combination on P21 gene expression in tumor cells PE/CA-PJ49 compared to normal cells HUVEC. Each sample was performed in duplicate. The samples were analyzed using the formula 2-ΔΔCt = gene expression. (* p < 0.05, ** p < 0.005; *** p < 0.0005).

Article Snippet: DuoSetR IC ELISA-Human Total p21/CIP1/CDKN1A(: DYC1047-2) was purchased from R&D Systems Inc. and contains the basic components required for the development of sandwich ELISAs to measure human p21 protein also known as CIP1 and CDKN1A in cell lysates.

Techniques: Gene Expression

Journal: Journal of Biological Chemistry

Article Title: Epidermal Growth Factor Receptor Pathway Analysis Identifies Amphiregulin as a Key Factor for Cisplatin Resistance of Human Breast Cancer Cells

doi: 10.1074/jbc.m706287200

Figure Lengend Snippet: FIGURE 3. Analysis of AKT kinase, downstream signaling reveals inactiva- tion of the p53 pathway in MCF-7 CisR cells. A, quantification of AKT kinase activity. To measure AKT kinase activity, a solid phase ELISA, which utilizes a specific synthetic peptide as a substrate and a polyclonal antibody that rec- ognizes the phosphorylated form of the substrate, was used. (n 3, ***, p 0.001). B, detection of p53 protein by immunoblotting (IB) using a polyclonal affinity-purified goat Ab specific for p53. MCF-7 cells (lane 1) and MCF-7 CisR cells (lane 2). Lane M, molecular weight marker. p53 is indicated by an arrow. C, quantification of p53 by a sandwich ELISA that measures human total p53 in cell lysates (n 3, ***, p 0.001). D, p21 expression indicates p53 pathway activity. Detection of p21 in whole cell lysates of MCF-7 (lane 1) and MCF-7 CisR cells (lane 2) by immunoblotting using a polyclonal affinity-purified goat Ab specific for p21. Lane M, molecular weight marker. p21 indicated by an arrow. E, to quantify the levels of p21 protein a sandwich ELISA that measures p21 in cell lysates was used. (n 3, ***, p 0.001). F, to quantify BCL-2 expression an ELISA that detects human BCL-2 in cell lysates was used (n 3, ***, p 0.001).

Article Snippet: For immunoblotting we used a polyclonal affinity-purified goat Ab specific for p53 at a concentration of 1 g/ml (AF1355, R & D Systems, Wiesbaden, Germany) and a polyclonal affinity-purified goat Ab specific for p21 at a concentration of 1 g/ml (AF1047, R & D Systems, Wiesbaden, Germany).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Affinity Purification, Molecular Weight, Marker, Sandwich ELISA, Expressing