primary antibodies p19arf  (Novus Biologicals)

Journal: bioRxiv

Article Title: Serum Amyloid P Secreted by Bone Marrow Adipocytes Drives Skeletal Amyloidosis

doi: 10.1101/2024.08.15.608092

Figure Lengend Snippet: ( A-C ) Tibial bones were harvested from 9-month-old APP-PS1 mice (AD) and their wild-type littermates (WT). Bone tissue sections were subjected to RNAscope assays using specific probes to p19Arf and H2AX . Representative images are shown in figure ( A ). The quantification of p19Arf + mean intensity and H2AX + mean intensity was shown in ( B ) and ( C ), respectively. n= 6. ( D-F ) Tibial bones were harvested from 24-month-old C57BL/6 mice (Old) compared to 4-month- old C57BL/6 mice (Young). Bone tissue sections were subjected to RNAscope assays using specific probes to p19Arf and H2AX . Representative images are shown in figure ( D ). Quantification of p19Arf + mean intensity and H2AX + mean intensity is shown in ( E ) and ( F ), respectively. Scale bar = 100 pm. n= 6. ( G-J ) Double-immunofluorescence staining of tibial bone sections from APP-PS1 mice (AD), and wild-type littermates (WT) was performed using antibodies against Perilipin and p19Arf or γH2AX. Representative images of Perilipin + p19Arf + cells from secondary spongiosa area of tibia are shown in ( G ), and analysis of cell number per mm 2 tissue area (N. Perilipin + p19Arf + cells) are shown in ( H ). Representative images of Perilipin+γH2AX + cells from secondary spongiosa area of tibia are shown in ( I ), and analysis of cell number per mm 2 tissue area (N. Perilipin + γH2AX + cells) are shown in ( J ). Scale bar = 200 pm. n=6. ( K-L ) Double-immunofluorescence staining of tibia bone sections from 24-month-old mice (Old) and 4-month-old mice (Young) was performed using antibody against Perilipin and γH2AX. Representative images of Perilipin + p19Arf + cells from secondary spongiosa area of tibia are shown in ( K ), and analysis of cell number per mm2 tissue area (N. Perilipin + γH2AX + cells) are shown in ( L ). Scale bar= 200 pM. n=6. ( M-Q ) Chromatin immunoprecipitation (ChIP)-qPCR assays. Schematic diagram of the p19Arf promoter with four CEBPα-binding sites (M). Chromatin immunoprecipitation (ChIP) assay on the promoter of p19Arf in isolated BMAds from APP-PS1 mice (AD) and wild-type littermates (WT) ( N-O ) and from 24-month-old mice (Old) and 4-month-old mice (Young) ( P-Q). CEBPα antibody or IgG antibody (negative control) were used for the ChIP assay. ChIP and input DNA were measured using RT-qPCR. RNA poly II was included as a positive control. Representative agarose gel pictures are shown in ( N ) and (P ). Quantitively fold enrichment ratios for DNA immunoprecipitated by CEBPα at different binding sites are shown in (O) and (Q). Data are represented as mean ±s.e.m. ***p<0.001, ****p<0.0001, as determined by unpaired two-tailed Student’s t-tests for 2 groups.

Article Snippet: Bone sections were blocked in PBS with 3% BSA for 1 hour and then stained overnight (>8 hours) with individual primary antibodies p19Arf (Novus NB200-111, 1:100), γH2AX (Cell signaling, 20E3, 1:200), Perilipin (Cell signaling, 9349, 1:200), Perilipin (Sigma, P1873, 1:500), and H31L21 (Thermo Fisher, Scientific 700254, 1:100).

Techniques: RNAscope, Double Immunofluorescence Staining, Chromatin Immunoprecipitation, Binding Assay, Isolation, Negative Control, Quantitative RT-PCR, Positive Control, Agarose Gel Electrophoresis, Immunoprecipitation, Two Tailed Test

Journal: JCI Insight

Article Title: BPDCN MYB fusions regulate cell cycle genes, impair differentiation, and induce myeloid–dendritic cell leukemia

doi: 10.1172/jci.insight.183889

Figure Lengend Snippet: ( A ) V5 and IgG CUT&RUN tracks at the Il3ra , Bcl2 , and Myc loci in Cdkn2a -KO MYB::PLEKHO1 leukemia cells. ( B ) TPM of Il3ra (CD123), Bcl2 , Myc , Ncam1 (CD56), Nrp1 (BDCA4), and Irf4 in Hoxb8-FL cell–derived pDCs ( n = 8) and Hoxb8-FL cell–derived leukemias of the indicated genotypes ( n = 4 per genotype). ( C ) Growth curves of Hoxb8-FL.MS5 cells transduced with V5-MYB constructs of the indicated genotypes. ( D ) Western blots showing p16INK4A and p19ARF expression in Hoxb8-FL.MS5 cells of the indicated genotypes. ( E ) Cell cycle analysis of Hoxb8-FL.MS5 cells. (Left) Representative flow plots of the indicated genotypes. (Right) Quantification of cells in each cell cycle phase as a proportion of total cells ( n = 4 per genotype). Significance tests compare proportion of cells in G1. ( A and E ) Data represent mean ± SEM. Significance determined by 1-way ANOVA with Tukey correction for multiple comparisons. * P <0.05, ** P <0.01, *** P < 0.001.

Article Snippet: Antibodies used for Western blots were: MYB (Abcam, ab45150), V5 (Cell Signaling Technology, 13202), ACTB (Santa Cruz Biotechnology Inc., sc-47778), GAPDH (Cell Signaling Technology, 2118), p16INK4A (Cell Signaling Technology, 29271), and p19ARF (Cell Signaling Technology, 77184).

Techniques: Derivative Assay, Transduction, Construct, Western Blot, Expressing, Cell Cycle Assay

Journal: Aging

Article Title: Roles of pigment epithelium-derived factor in exercise-induced suppression of senescence and its impact on lung pathology in mice.

doi: 10.18632/aging.205976

Figure Lengend Snippet: Figure 1. Myocyte-derived factors suppress cellular senescence. (A) Wild-type MEFs were cultured according to the 3T3 protocol in the presence of a control medium or C2C12-CM (1:1 dilution with DMEM containing 10% serum). Data represent the average value of triplicate samples ± SD. (B, C) Total RNA was isolated from cells and the expression of Ink4a (B) or Arf (C) was analyzed by real-time PCR. Values were normalized to Gapdh in each sample. (D) The expression of p16INK4a and p21 was analyzed by immunoblotting. β-Actin was used as a loading control. (E) An immunofluorescence analysis was performed using the p19ARF antibody. Cells were counterstained with DAPI. Scale bar, 100 μm. (F) The number of p19ARF-positive cells was counted in (E). (G) Cells (passage 4) were stained for SA-β-gal. Scale bar, 100 μm. (H) The number of SA-β-gal-positive cells was counted in each sample. Scale bar, 100 μm. Values represent means ± SD. Data were analyzed by a one-way ANOVA and Tukey’s post-hoc analysis (B, C, F) or the Student’s t-test (H). *P <0.05, **P <0.01, and ***P <0.001.

Article Snippet: Proteins were detected with antibodies to p19ARF, p21, BCL-6, phospho-ERK1/2 (sc-32748, sc6246, sc-7388, and sc-136521; all from Santa Cruz Biotechnology, Inc.), β-Actin, β-catenin, phospho-Akt (ser473), phospho-p38 (#12620, #9562, #4060, and #9211; all from Cell Signaling Technology, Inc.), adipose triglyceride lipase (ATGL) (55190-1-AP, Proteintech, Inc.), and p16INK4a [68].

Techniques: Derivative Assay, Cell Culture, Control, Isolation, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining

Journal: Aging

Article Title: Roles of pigment epithelium-derived factor in exercise-induced suppression of senescence and its impact on lung pathology in mice.

doi: 10.18632/aging.205976

Figure Lengend Snippet: Figure 2. PEDF mediates the anti-cellular senescence effects of C2C12-CM. (A–C) MEFs were cultured in the presence of C2C12-CM treated with a control or PEDF antibody for 3 days. (A) Cell numbers were counted and relative changes in cell numbers in 3 days were plotted. (B) Total RNA was isolated from MEFs and the expression of Ink4a and Arf was analyzed by real-time PCR. Values were normalized to Gapdh in each sample. (C) p16INK4a and p19ARF levels were analyzed by immunoblotting. β-Actin was used as the loading control. (D) MEFs were cultured in the presence of a recombinant of PEDF (100 ng/mL) for 3 days. Changes in cell numbers were plotted. (E) Cell viability was determined by the trypan blue exclusion assay. (F) The expression of Ink4a, Arf, and p21 was analyzed by real-time PCR. Values were normalized to Gapdh in each sample. (G) p16INK4a, p19ARF, and p21 levels were analyzed by immunoblotting. β-Actin was used as a loading control. (H) Cells were stained for SA-β-gal. Scale bar, 100 μm. (I) The percentage of SA-β-gal-positive cells was plotted. (J) Cells were stimulated with the indicated concentrations of recombinant PEDF for 3 days. Intracellular ROS levels were analyzed in each sample, and relative values were plotted against the average of the control sample. Values represent means ± SD. Data were analyzed by the Student’s t-test (A, B, D–F, I) or a one-way ANOVA and Tukey’s post-hoc analysis (J). *P <0.05, **P <0.01, and ***P <0.001.

Article Snippet: Proteins were detected with antibodies to p19ARF, p21, BCL-6, phospho-ERK1/2 (sc-32748, sc6246, sc-7388, and sc-136521; all from Santa Cruz Biotechnology, Inc.), β-Actin, β-catenin, phospho-Akt (ser473), phospho-p38 (#12620, #9562, #4060, and #9211; all from Cell Signaling Technology, Inc.), adipose triglyceride lipase (ATGL) (55190-1-AP, Proteintech, Inc.), and p16INK4a [68].

Techniques: Cell Culture, Control, Isolation, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Recombinant, Trypan Blue Exclusion Assay, Staining