p p53 ser15  (Proteintech)

Journal: bioRxiv

Article Title: Single-cell full-length transcriptome of human lung reveals genetic effects on isoform regulation beyond gene-level expression

doi: 10.64898/2026.03.27.714873

Figure Lengend Snippet: ( A ) Pie chart presents the fraction of GWAS-colocalized eIsoforms with an eQTL-colocalizing isoQTL for a matched eGene in the same cell type. ( B ) Pie chart presents the fraction of TWAS significant isoforms overlapping TWAS genes from cell-barcode matched short-read eQTL data ( C ) isoQTL or eQTL colocalization with lung cancer GWAS at the PPIL6 locus in multiciliated cells. SNPs are color-coded based on the LD R 2 (1000 Genomes, EAS, phase 3) with isoQTL lead SNP, rs12528822 (purple diamond). ( D ) Association between the genotype of lead isoQTL rs12528822 and the normalized expression of PPIL6-207 (top panel) and TALONT003040002 (bottom panel). ( E ) Association between the genotype of lead isoQTL rs12528822 and the proportion of PPIL6-207 (top panel) and TALONT003040002 (bottom panel) over all PPIL6 isoforms. P -value is calculated by linear regression with the same covariates used in isoQTL mapping adjusted. Allele C of rs12528822 is the risk-associated allele for lung cancer. The violins and boxes are colored by genotype, and the grey line shows the trend of association. Center lines show the medians; the box indicates the middle of 50% of data; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; outliers are represented by dots; density of normalized expression is represented by the width of violin shape. ( F , H ) Representative histograms of DNA damage marker γH2AX ( F ) or p-p53 ( H ) for overproduced PPIL6 isoforms or HPS4 in GFP + transfected cells from the same experimental batch. ( G,I ) Bar plots showed the DNA damage level (i.e., γH2AX or p-p53) normalized to the median intensity of GFP + HPS4-overproducing MRC5-SV40 cells. Error bars show the mean±standard error. Black dots show the individual level of normalized value of two replicates from three experiments (n = 6). One-way ANOVA is used to test the differences across groups, * represents adjusted p- value < 0.05 by post hoc Tukey’s test. All summary statistics are in Table S11 .

Article Snippet: Antibody staining was performed using anti–phospho–histone H2A.X (Ser139) antibody (clone JBW301; 1:750; Sigma–Aldrich, 05-636-25UG) and p-p53 (Ser15) mouse monoclonal antibody (clone 16G8; 1:1000; Cell Signaling Technology, 9286), and Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa FluorTM 647 (1:1000 dilution; Invitrogen, A-21235).

Techniques: Expressing, Marker, Transfection

Journal: Current Issues in Molecular Biology

Article Title: MOB2 Loss Sensitizes Lung Cancer Cells to PARP Inhibition Through p53-Dependent DNA Damage Signaling

doi: 10.3390/cimb48030295

Figure Lengend Snippet: p53 is required for hMOB2-dependent sensitization to PARP inhibition in lung cancer cells. ( A ) Representative immunoblot analysis of phospho- p53 , hMOB2 and loading control ( GAPDH ) in A549 cells transfected with control (siCTRL) or hMOB2-targeting siRNA (siMOB2) and treated with vehicle (DMSO) or olaparib (20 μM) for 72 h ( n = 2). ( B , C ) Densitometric analysis of the immunoblot shown in Panel ( A ). ( D ) Immunoblot validation of stable p53 reconstitution in the p53-null H1299 lung cancer cell line following retroviral transduction with empty vector (EV) or wild-type p53-expressing constructs. Two independent p53-expressing populations are shown. Actin serves as a loading control ( n = 1). ( E , F ) Densitometric analysis of the immunoblot shown in Panel ( D ). ( G ) Immunoblot confirmation of hMOB2 knockdown in p53-null or -reconstituted H1299 cells (replicate #2) following transfection with control (siCTRL) or hMOB2-targeting siRNA (siMOB2). Actin serves as a loading control ( n = 1). ( H , I ) Densitometric analysis of the immunoblot shown in Panel ( G ). ( J ) Representative clonogenic survival images of p53-null or -reconstituted H1299 cells transfected with siCTRL or siMOB2 and treated with vehicle (DMSO) or olaparib (20 μM) for 8–12 days (1000 cells were seeded per well). ( K ) Quantification of clonogenic survival shown in ( C ), expressed as surviving fraction relative to untreated controls. Data represent mean ± SEM from at least three independent experiments. Statistical significance was assessed using unpaired Student’s t -test. p < 0.05 (*), p < 0.01 (**). ns : non-significant.

Article Snippet: For detection of proteins, including hMOB2 (home-made, [ ]), p-p53 (S15) (Cell Signaling Technology, Danvers, MA, USA, #9284), p21 (Cell Signaling Technology, Danvers, MA, USA, #2947), tubulin (home-made), actin (Santa Cruz, Dallas, TX, USA, #sc-1616), GAPDH (Santa Cruz, Dallas, TX, USA, #sc-365062), membranes were blocked and incubated in TBST containing 5% bovine serum albumin (BSA) to minimize non-specific binding.

Techniques: Inhibition, Western Blot, Control, Transfection, Biomarker Discovery, Retroviral, Transduction, Plasmid Preparation, Expressing, Construct, Knockdown

Journal: Nucleic Acids Research

Article Title: REV7 associates with ATRIP and inhibits ATR kinase activity

doi: 10.1093/nar/gkaf1527

Figure Lengend Snippet: REV7 inhibits ATR activity. ( A ) REV7 disassociated p53 fragment (2–50) affects ATR kinase activity in vitro . 293T cells were transfected with Flag-tagged ATR (F-ATR) or empty vector control. Cells were harvested 48 h after transfection, and immunoprecipitation was performed using M2 agarose beads. The beads were washed twice with kinase buffer. 10 μl of buffer or 10 μg of His-REV7 were added, and samples were incubated at 30°C for 15 min. 10 μl of reaction buffer containing substrate (purified GST-p53 fragment) was added, followed by a 30 min incubation at 30°C. After electrophoretic transfer of proteins, the membrane was immunoblotted with the indicated antibodies. Results for the immunoprecipitation (IP) product after gel electrophoresis are shown. The shifted P-p53 Ser15 bands (**) and (*) are the same size as the shifted GST-p53 (2–50) (**) and GST-p53 (2–50) (*) bands, respectively. ( B ) western blot of DDR activation following a 24-h 40 ng/ml mitomycin C (MMC), 40 μM hydroxyurea (HU), or mock exposure in TK6 (REV7+/+), MB86 (REV7-/-), and MB92 (REV7-/-) cells. Protein loading was equalized based on cell number. After the electrophoretic transfer of proteins, the western blot membranes were immunoblotted with the appropriate antibodies. ( C ) The increase in phospho-CHK1 (Ser317) (P-CHK1 S317) protein was rescued when we complemented REV7-/- cells with a REV7 expression vector. Protein loading was equalized based on cell number. Expression of CHK1, phospho-CHK1 (Ser317), phospho-CHK1 (Ser345) (P-CHK1 S345), phospho-p53 (Ser15) ( P -53 S15), p53, αTubulin, REV7, ATR, and ATRIP proteins was determined by western blot in TK6 (REV7+/+) with empty vector and MB86 (REV7-/-) cells with empty vector or REV7-FH. (D-E) western blot of DDR activation following a 24-h 40 ng/ml mitomycin C (MMC), 40 μM hydroxyurea (HU), or mock exposure in TK6, REV3L-/- ( D ) or 53BP1-/- ( E ) TK6 cells. Protein loading was equalized based on cell number. After the electrophoretic transfer of proteins, the western blot membranes were immunoblotted with the appropriate antibodies. See also and .

Article Snippet: P-p53 Ser15 (9284), 1:1 000; P-ATR Thr1989 (30 632), 1:1 000; P-Chk1 Ser345 (2348), 1:1 000; monoclonal anti-HA (3724), 1:1 000; and P-Chk1 Ser317 (12 302), 1:1 000, were obtained from Cell Signaling.

Techniques: Activity Assay, In Vitro, Transfection, Plasmid Preparation, Control, Immunoprecipitation, Incubation, Purification, Membrane, Nucleic Acid Electrophoresis, Western Blot, Activation Assay, Expressing