goat anti-p21  (R&D Systems)

Journal: Bone & Joint Research

Article Title: eIF5A downregulated by mechanical overloading delays chondrocyte senescence and osteoarthritis by regulating the CREBBP-mediated Notch pathway

doi: 10.1302/2046-3758.142.bjr-2024-0288.r1

Figure Lengend Snippet: Fig. 1 Eukaryotic translation initiation factor 5A (eIF5A) is reduced in articular cartilage in osteoarthritis (OA) patients and OA mice. a) Volcano plots of differentially expressed genes after stretching. b) Quantitative polymerase chain reaction (qPCR) analysis of eIF5A, type II collagen (COL2), matrix metalloproteinase 13 (MMP13), P21, and P16 in primary chondrocytes treated with 20% cyclic tensile strain for 24 hours; n = 6 per group. c) Western blot (WB) analysis of eIF5A, COL2, and MMP13 in primary chondrocytes treated with 20% cyclic tensile strain for 24 hours. Glyceraldehyde-3- phosphate dehydrogenase (GAPDH) was used as the loading control. d) Safranin O/Fast Green staining (upper) and immunohistochemistry (IHC) staining of eIF5A (lower) of human knee cartilage from the medial (OA) and lateral (controls) tibial plateau of OA patients. Scale bar: 100 µm. e) Safranin O/Fast Green staining (upper) and IHC of eIF5A (lower) of control group (CON) and destabilization of the medial meniscus (DMM) knee cartilage of C57 mouse. Knee tissue was obtained at four and eight weeks (4W and 8W) after the DMM procedure. Scale bars: 100 µm, 50 µm. f) Osteoarthritis Research Society International (OARSI) grades for human articular cartilage of the medial and lateral tibial plateau in Fig. 1c and quantification of eIF5A in each group; n = 6 per group. g) OARSI grades for the joints described in Fig. 1e and quantification of eIF5A in each group; n = 6 per group. An independent-samples t-test and one-way analysis of variance were used for statistical analyses (*p < 0.05, **p < 0.01, ***p < 0.001). FC, fold change; NC, negative control; ns, not significant.

Article Snippet: The following primary antibodies were used for this Western blot (WB) analysis: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10,000, HA721136; HUABIO, USA), eIF5A eIF5A downregulated by mechanical overloading delays chondrocyte senescence J. Huang, J. Zheng, J. Yin, et al 125 (1:1,000, A2016; ABclonal, Germany), MMP13 (1:1,000, A11148; ABclonal), type II collagen (COL2) (1:1,000, A1560; ABclonal), P21 (1:1,000, ab109199; Abcam, UK), P16 (1:2,000, ab211542; Abcam), Notch intracellular domain (NICD) (1:1,000, 20687-1; Proteintech, USA), and CREBBP (1:1,000, A14237, ABclonal).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Control, Staining, Immunohistochemistry, Negative Control

Journal: Bone & Joint Research

Article Title: eIF5A downregulated by mechanical overloading delays chondrocyte senescence and osteoarthritis by regulating the CREBBP-mediated Notch pathway

doi: 10.1302/2046-3758.142.bjr-2024-0288.r1

Figure Lengend Snippet: Fig. 2 Knockdown/overexpression of eukaryotic translation initiation factor 5A (eIF5A) can aggravate or rescue the injury of chondrocytes in osteoarthritis (OA). a) Quantitative polymerase chain reaction (qPCR) analysis of eIF5A, type II collagen (COL2), martrix metalloproteinase 13 (MMP13), P21, and P16 in primary chondrocytes transfected with si-eIF5A; n = 6 per group. b) qPCR analysis of eIF5A, COL2, MMP13, P21, and P16 in mouse primary chondrocytes treated with or without OE-eIF5A under 20% cyclic tensile strain; n = 6 per group. c) Western blot (WB) analysis and quantification of eIF5A, COL2, MMP13, P21, and P16 in si-eIF5A-transfected primary chondrocytes; n = 3 per group. d) WB analysis and quantification of eIF5A, COL2, MMP13, P21, and P16 in mouse primary chondrocytes treated with or without OE-eIF5A at 20% cyclic tensile strain; n = 3 per group. e) and f) Immunofluorescence (IF) and senescence-associated β-galactosidase (SA-βGal) staining of eIF5A after transfection of primary chondrocytes with si-eIF5A and corresponding quantitative plots; n = 3 per group. g) and h) IF and SA-βGal staining of primary mouse chondrocytes treated with or without OE-eIF5A at 20% cyclic tensile strain and corresponding quantification plots; n = 3 per group. An independent-samples t-test and one-way analysis of variance were used for statistical analyses (*p < 0.05, **p < 0.01, ***p < 0.001). CON, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NC, negative control; ns, not significant; OE, over-expression.

Article Snippet: The following primary antibodies were used for this Western blot (WB) analysis: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10,000, HA721136; HUABIO, USA), eIF5A eIF5A downregulated by mechanical overloading delays chondrocyte senescence J. Huang, J. Zheng, J. Yin, et al 125 (1:1,000, A2016; ABclonal, Germany), MMP13 (1:1,000, A11148; ABclonal), type II collagen (COL2) (1:1,000, A1560; ABclonal), P21 (1:1,000, ab109199; Abcam, UK), P16 (1:2,000, ab211542; Abcam), Notch intracellular domain (NICD) (1:1,000, 20687-1; Proteintech, USA), and CREBBP (1:1,000, A14237, ABclonal).

Techniques: Knockdown, Over Expression, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Immunofluorescence, Staining, Control, Negative Control

Journal: Bone & Joint Research

Article Title: eIF5A downregulated by mechanical overloading delays chondrocyte senescence and osteoarthritis by regulating the CREBBP-mediated Notch pathway

doi: 10.1302/2046-3758.142.bjr-2024-0288.r1

Figure Lengend Snippet: Fig. 3 Eukaryotic translation initiation factor 5A (eIF5A) can inhibit cartilage degeneration and delay osteoarthritis (OA) progression in destabilization of the medial meniscus (DMM)-OA mice. a) Protocol for intra-articular injection of ADV-NC or ADV-eIF5A in medial meniscus instability (DMM) mice. b) Analysis of weekly weightbearing asymmetry in mice. c) Safranin O/Fast Green staining of knee cartilage from control, DMM, and post-DMM eIF5A adenovirus-injected mice. Scale bar: 100 µm. d) and e) Immunohistochemistry (IHC) staining of eIF5A and type II collagen (COL2) in knee cartilage of control, DMM, and post-DMM eIF5A adenovirus-injected mice. Scale bar: 50 µm. f) to h) Immunofluorescence (IF) staining of matrix metalloproteinase (MMP13), P16, and P21 in knee cartilage of control, DMM, and post-DMM eIF5A adenovirus-injected mice. Scale bar: 50 µm. i) to n) Quantitative analysis of the Osteoarthritis Research Society International (OARSI) scoring scale in mice in Fig. 3a and the number of positive particles in Fig. 3d to 3h of articular cartilage; n = 6 per group. One-way analysis of variance was used for statistical analyses (*p < 0.05, **p < 0.01, ***p < 0.001). 4W, four weeks; ADV, adenovirus; NC, negative control; ns, not significant.

Article Snippet: The following primary antibodies were used for this Western blot (WB) analysis: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10,000, HA721136; HUABIO, USA), eIF5A eIF5A downregulated by mechanical overloading delays chondrocyte senescence J. Huang, J. Zheng, J. Yin, et al 125 (1:1,000, A2016; ABclonal, Germany), MMP13 (1:1,000, A11148; ABclonal), type II collagen (COL2) (1:1,000, A1560; ABclonal), P21 (1:1,000, ab109199; Abcam, UK), P16 (1:2,000, ab211542; Abcam), Notch intracellular domain (NICD) (1:1,000, 20687-1; Proteintech, USA), and CREBBP (1:1,000, A14237, ABclonal).

Techniques: Injection, Staining, Control, Immunohistochemistry, Immunofluorescence, Negative Control

Journal: Bone & Joint Research

Article Title: eIF5A downregulated by mechanical overloading delays chondrocyte senescence and osteoarthritis by regulating the CREBBP-mediated Notch pathway

doi: 10.1302/2046-3758.142.bjr-2024-0288.r1

Figure Lengend Snippet: Fig. 4 Intra-articular injection of eukaryotic translation initiation factor 5A (eIF5A) partially relieved the progression of 13.5 N overload-induced osteoarthritis (OA). a) Protocol for intra-articular injection of ADV-NC or ADV-eIF5A in mice after stimulation with an excessive mechanical load of 13.5 N (13.5 N model). b) Analysis of weekly weightbearing asymmetry in mice. c) Safranin O/Fast Green staining of knee cartilage from control, 13.5 N, and post-13.5 N eIF5A adenovirus injected mice. Scale bar: 100 µm. d) and e) Immunohistochemistry (IHC) staining of eIF5A and type II collagen (COL2) in knee cartilage of control, 13.5 N group, and post-13.5 N eIF5A adenovirus-injected mice. Scale bar: 50 µm. f) to h) Immunofluorescence (IF) staining for matrix metalloproteinase (MMP13), P16, and P21 in knee cartilage of control, 13.5 N group, and post-13.5 N eIF5A adenovirus-injected mice. Scale bar: 50 µm. i) to n) Quantitative analysis of the Osteoarthritis Research Society International (OARSI) scoring scale in mice in Fig. 4a and the number of positive particles in Fig. 4d to 4h of articular cartilage; n = 6 per group. One-way analysis of variance was used for statistical analyses (*p < 0.05, **p < 0.01, ***p < 0.001). ADV, adenovirus; NC, negative control; ns, not significant.

Article Snippet: The following primary antibodies were used for this Western blot (WB) analysis: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10,000, HA721136; HUABIO, USA), eIF5A eIF5A downregulated by mechanical overloading delays chondrocyte senescence J. Huang, J. Zheng, J. Yin, et al 125 (1:1,000, A2016; ABclonal, Germany), MMP13 (1:1,000, A11148; ABclonal), type II collagen (COL2) (1:1,000, A1560; ABclonal), P21 (1:1,000, ab109199; Abcam, UK), P16 (1:2,000, ab211542; Abcam), Notch intracellular domain (NICD) (1:1,000, 20687-1; Proteintech, USA), and CREBBP (1:1,000, A14237, ABclonal).

Techniques: Injection, Staining, Control, Immunohistochemistry, Immunofluorescence, Negative Control

Journal: Bone & Joint Research

Article Title: eIF5A downregulated by mechanical overloading delays chondrocyte senescence and osteoarthritis by regulating the CREBBP-mediated Notch pathway

doi: 10.1302/2046-3758.142.bjr-2024-0288.r1

Figure Lengend Snippet: Fig. 6 Eukaryotic translation initiation factor 5A (eIF5A) delays osteoarthritis (OA) through inhibiting cyclic-adenosine monophosphate (AMP) response element binding protein (CREB)-binding protein (CREBBP) expression. a) Western blot (WB) analysis of Notch intracellular domain (NICD), CREBBP, type II collagen (COL2), matrix metalloproteinase (MMP13), P21, and P16 in primary mouse chondrocytes treated with or without si-CREBBP at 20% cyclic tensile strain. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the loading control. b) The WB analysis of NICD, CREBBP, COL2, MMP13, P21, P16, and eIF5A in mouse primary chondrocytes treated with or without OE-eIF5A after OE-CREBBP. GAPDH was used as the loading control. c) and d) Quantification of the expression of each indicator in Fig. 6a and 6b; n = 3 per group. e) and f) Senescence-associated β-galactosidase (SA-βGal) staining of primary mouse chondrocytes treated with or without si-CREBBP at 20% elongation strain loading and corresponding quantification plots; n = 3 per group. g) and h) SA-βGal staining of primary mouse chondrocytes treated with or without OE-eIF5A after OE-CREBBP and corresponding quantification plots; n = 3 per group. One-way analysis of variance was used for statistical analyses (*p < 0.05, **p < 0.01, ***p < 0.001). CON, control; ns, not significant.

Article Snippet: The following primary antibodies were used for this Western blot (WB) analysis: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10,000, HA721136; HUABIO, USA), eIF5A eIF5A downregulated by mechanical overloading delays chondrocyte senescence J. Huang, J. Zheng, J. Yin, et al 125 (1:1,000, A2016; ABclonal, Germany), MMP13 (1:1,000, A11148; ABclonal), type II collagen (COL2) (1:1,000, A1560; ABclonal), P21 (1:1,000, ab109199; Abcam, UK), P16 (1:2,000, ab211542; Abcam), Notch intracellular domain (NICD) (1:1,000, 20687-1; Proteintech, USA), and CREBBP (1:1,000, A14237, ABclonal).

Techniques: Binding Assay, Expressing, Western Blot, Control, Staining

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Schistosoma mansoni-Induced Oxidative Stress Triggers Hepatocellular Proliferation.

doi: 10.1016/j.jcmgh.2023.08.014

Figure Lengend Snippet: Figure 2. S. mansoni infection triggered the replication stress response. (A) Gene expression (mRNA level) of checkpoint kinase 1 (Chek1) was up-regulated in livers of bs-infected hamsters (n ¼ 5–8). Data were normalized to the control group, and the Kruskal–Wallis test was performed to assess group differences. (B and C) Phosphorylation of CHK1 at Ser345 revealed checkpoint activation in livers of bs-infected hamsters as determined by Western blot analysis and densitometric analysis. (B and D) The tumor-suppressor protein p53 was up-regulated in livers of bs-infected hamsters as determined by Western blot analysis and densitometric analysis. (B and E) PCNA was increased at the protein level in livers of bs-infected hamsters. (F) The mRNA expression levels of cyclin-dependent kinase inhibitor p21SDI1 (Cdkn1a), essential for DNA damage response, were increased in livers of bs-infected hamsters (n ¼ 5–8). (G) Representative co-immunostainings for p21SDI1 (red)/fibrinogen (grey) in the livers of female hamsters, which were noninfected (control), ss-infected, and bs-infected. The acute-phase protein fibrinogen is synthesized in hepatocytes and here stained the cytoplasm of hepatocytes light grey. This allows identification of parenchymal p21 expression (arrows) in hepatocytes. Arrows indicate p21 staining in hepatocellular cytoplasm, while ar- rowheads point out p21-stained nonparenchyma cells. Of note, p21SDI1 protein was detected mainly in the cytoplasm of hepatocytes in bs-infected hamsters. Original magnification: 200 and 1000. Scale bars: 100 and 25 mm. *Eggs. Dashed line indicates the granuloma border. Representative Western blots are depicted. Data were normalized to the control group, and the Kruskal–Wallis test was performed to assess group differences. Levels of significance are indicated in the figure. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ni, noninfected; p, portal tract; pCHK1, phosphorylated checkpoint kinase 1.

Article Snippet: After heat-induced epitope retrieval, using citrate buffer, the slides were incubated with polyclonal rabbit antifibrinogen (27913; Abcam) in combination with polyclonal goat antiMCM6 (9843; Santa Cruz) and polyclonal goat anti-p21 (397G; Santa Cruz).

Techniques: Infection, Gene Expression, Control, Phospho-proteomics, Activation Assay, Western Blot, Expressing, Synthesized, Staining

Journal: Aging (Albany NY)

Article Title: Increased expression of musashi 1 on breast cancer cells has implication to understand dormancy and survival in bone marrow

doi: 10.18632/aging.204620

Figure Lengend Snippet: Cell cycle-associated gene levels in Msi 1 knockdown BCCs. ( A ) Shown is the pathway tested by Western blots. ( B ) Western blots were performed with whole cell extracts for p21, p16 in the same blot and separate blots for p15 and p53. ( C ) Western blots were performed for Bcl2 and CDK4 in the same blot and separate blots for Cyclin E and p-Rb. Each membrane was stripped and reprobed for β-actin. The blots represent the mean±SD of three independent experiments. ( D – G ) Normalized bands are shown for key proteins in the Western blots, ±SD, n=3. ( H ) A summary of the cycling protein levels relative to the cycling phase.

Article Snippet: Goat anti-p21 was purchased from R&D Systems (Minneapolis, MN), goat anti-Oct4a, and rabbit anti-Msi-1 from Abcam (Cambridge, MA); mouse anti-Hes1 from NovusBio (Centennial, Colorado); rabbit anti-p16, rabbit anti-phospho-PTEN, mouse anti-Rb, rabbit anti-phospho-PDK1, rabbit anti-PTEN, rabbit anti-PD-L1 and mouse anti-Cyclin E from Cell Signaling Technology (Massachusetts, USA).

Techniques: Knockdown, Western Blot, Membrane

Journal: Cell reports

Article Title: Sex-biased islet β cell dysfunction is caused by the MODY MAFA S64F variant by inducing premature aging and senescence in males

doi: 10.1016/j.celrep.2021.109813

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Goat anti-P21 (for cells) , Santa Cruz , Cat#sc-397G; RRID:AB_632127.

Techniques: Virus, Modification, Recombinant, Staining, Software