Journal: PLoS ONE

Article Title: The p53 R172H Mutant Does Not Enhance Hepatocellular Carcinoma Development and Progression

doi: 10.1371/journal.pone.0123816

Figure Lengend Snippet: A) Immunoblot demonstrating knockdown of p53 in cell lines derived from p53 R172H HCCs as compared to pGIPZ empty vector control infections. B) Quantitative RT-PCR analysis of p53 family transcriptional targets in four mouse HCC cell lines with knockdown of p53 R172H . Ct values in each sample were normalized to β-Actin as an endogenous reference. Fold changes were calculated by normalizing all samples to the average Ct value of the cell line’s pGIPZ control. The Comparative Ct method was used for fold change calculation. C) Immunoblots demonstrating the expression of p63 and p73 isoforms in p53 fl/fl and p53 R172H HCC cell lines. Note the presence of multiple isoforms within the cell lines. GAPDH is used as a loading control. MW = molecular weight marker.

Article Snippet: Antibodies used were: p53 antibody (1:2000, Cell Signaling 1C12), p21 antibody (1:1000, Santa Cruz sc-6246), TAp63 antibody (1:500, Biolegend 618901), ΔNp63 antibody (1:500, Biolegend 619001), Total p63 antibody (1:500 Santa Cruz, ac-8431), TAp73 antibody (1:500 Novus 24737), Total p73 antibody (1:500 Imgenex).

Techniques: Western Blot, Derivative Assay, Plasmid Preparation, Quantitative RT-PCR, Expressing, Molecular Weight, Marker

Journal: American Journal of Physiology - Cell Physiology

Article Title: Protein kinase G increases antioxidant function in lung microvascular endothelial cells by inhibiting the c-Abl tyrosine kinase

doi: 10.1152/ajpcell.00375.2012

Figure Lengend Snippet: A: wild-type mouse lung microvascular endothelial cells (MLMVEC). Representative Western blot showing the effect of 0, 250, and 500 μM H2O2 on phospho-Y245 c-Abl and total c-Abl in the presence and absence of 8pCPT-cGMP. Graph shows effect of 30-min 8pCPT-cGMP (50 μM) on H2O2-induced c-Abl activation in wild-type MLMVEC, as measured by c-Abl Y245 phosphorylation, controlled for total c-Abl (n = 5; *P < 0.05, significant interaction by ANOVA). B: wild-type MLMVEC. Representative Western blot showing the effect of 0, 250, and 500 μM H2O2 on phospho-Y99 p73 and total p73 in the presence and absence of 8pCPT-cGMP. Graph shows effect of 30-min 8pCPT-cGMP (50 μM) on H2O2-induced p73 phosphorylation in wild-type MLMVEC, controlled for total p73 (n = 6; *P < 0.05 by ANOVA). C: protein kinase GI mice (PKGI−/−) MLMVEC. Representative Western blots showing phosphorylation of vasodilator-stimulated phosphoprotein (VASP) serine 235 after 8pCPT-cGMP treatment in wild-type but not PKGI−/− MLMVEC, and the effect of 0, 250, and 500 μM H2O2 on phospho-Y245 c-Abl and total c-Abl in the presence and absence of 8pCPT-cGMP. D, left: graph shows effect of 30-min 8pCPT-cGMP on H2O2-induced c-Abl activation in PKGI−/− MLMVEC (n = 4; P = not significant). Data are normalized to wild-type mean. D, right: graph shows baseline c-Abl activity, as measured by phospho-Y245 c-Abl/total c-Abl ratio in wild-type vs. PKGI−/− MLMVEC in the absence of 8pCPT-cGMP or H2O2 exposure (n = 9 and 4; *P < 0.01). All values are presented as means ± SE.

Article Snippet: Total p73 and total c-Abl antibodies were from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Western Blot, Activation Assay, Activity Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Protein kinase G increases antioxidant function in lung microvascular endothelial cells by inhibiting the c-Abl tyrosine kinase

doi: 10.1152/ajpcell.00375.2012

Figure Lengend Snippet: A: wild-type MLMVEC. Representative western blot showing the effect of 0, 250, and 500 μM H2O2 on phospho-Y245 c-Abl and total c-Abl in the presence and absence of atrial natriuretic peptide (ANP; 10 nM). Graph shows effect of 30-min ANP (10 nM) on H2O2-induced c-Abl activation in wild-type MLMVEC, as measured by c-Abl Y245 phosphorylation, controlled for total c-Abl (n = 7; *P < 0.05 by ANOVA). B: wild-type MLMVEC. Representative Western blots showing the effect of 0, 250, and 500 μM H2O2 on phospho-Y99 p73 and total p73 in the presence and absence of ANP. Graph shows effect of H2O2 on p73 phosphorylation, controlled for total p73 (n = 7; *P < 0.05 for effect of H2O2 in diluent-treated cells by ANOVA). The absence of phospho-p73 in ANP-treated cells (30 min, 10 nM) precluded quantification; signal represented as zero in graph.

Article Snippet: Total p73 and total c-Abl antibodies were from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Western Blot, Activation Assay

Journal: Oncogene

Article Title: DNp73alpha protects myogenic cells from apoptosis.

doi: 10.1038/sj.onc.1209321

Figure Lengend Snippet: Figure 1 (a) p73 isoforms are differentially regulated during muscle differentiation. Left panel: total cell lysates were prepared from proliferating C2C12 cells cultured in growth medium (GM: DMEM with 20% fetal bovine serum (FBS), 2 mM glutamine and antibiotics), from C2C12 cells grown to confluence in GM (Time 0) and from confluent C2C12 cell cultures grown in differentiating medium (DM: DMEM supplemented with 0.1% FBS, insulin, transferrin, and selenium for the indicated times. Immunoblotting was performed as described (Costanzo et al., 2002) using a pan-p73 mouse monoclonal antibody (Imgenex #1288). Right panel: quantification of DNp73a and TAp73a protein levels by densitometric analysis. Data are expressed as fold increase (mean7s.d.) from two independent experiments. (b) p73b protein levels are growth-factors dependent. Total cell lysates were obtained from C2C12 cells cultured as in (a). In the middle and right panels, additional extracts were prepared from C3H10T1/2 fibroblasts and C2C7 myoblasts cultured in standard medium plus 10% FBS, shifted at low density in 0.1 FBS for 24 h (starved) and restimulated with serum ( þ FBS). Immunoblots were analysed using a TAp73b-specific monoclonal antiboby (Upstate #GC15). (c) DNp73 transcripts are upregulated in differentiating C2C12 cells of the total RNA 1 mg was extracted from proliferating and differentiating C2C12 cells with the TRIzol reagent (GIBCO BRL) and RNA was reverse-transcribed and amplified by the Superscript One Step RT–PCR (Invitrogen) using oligonucleotide primers that specifically recognize either TA or DNp73 transcripts (PCR condition and oligonucleotides are available upon request). Amplification of GAPDH transcripts was used to normalize equal loading of each RNA sample. Right panel: densitometric analysis performed as in (a).

Article Snippet: Immunoblotting was performed as described (Costanzo et al., 2002) using a pan-p73 mouse monoclonal antibody (Imgenex #1288).

Techniques: Cell Culture, Western Blot, Reverse Transcription, One Step RT-PCR

Journal: Oncogene

Article Title: DNp73alpha protects myogenic cells from apoptosis.

doi: 10.1038/sj.onc.1209321

Figure Lengend Snippet: Figure 2 (a) DNp73 promoter activity is upregulated in myoblasts vs myotubes. C2C12 cells were transiently transfected using the calcium phosphate precipitation method with either the P2p73-luc (Vossio et al., 2002) or the P1p73-luc (Pediconi et al., 2003) reporter plasmids. Luciferase activity was determined at the indicated time. Error bars represent standard deviations from three independent experiments. (b) Schematic representation of the mouse P2p73 promoter. Putative p53 sites and E-box like sites are indicated. P2p73 E-box like elements: (I) 1941/1936; (II) 1770/1765; (III) 1750/1745; (IV) 1374/1369; (V) 355/350. P2p73p53 elements: (A) 1363/1331; (B) 1253/1219; (C) 1203/1183; (D) 307/298; (E) 62/32. Nucleotide positions are relative to the ATG present in the Exon30. (c and d) p53 and p73 regulate DNp73 promoter activity. C2C12 cells were transiently co-transfected with the P2p73-luc reporter in combination with either the indicated DN-expressing vectors (c) or the indicated siRNAs (Dharmacon.lnc) (d). pCDNAHA-p73DD expresses a p73 miniprotein that acts as a selective DN of p73-dependent, but not p53- dependent, transcription (Irwin et al., 2000). The p53DD plasmid is a gift from Dr M Oren. (e) ZEB inhibits the upregulation of the P2p73 promoter in differentiating myocytes. C2C12 cells were transiently transfected with the P2p73-luc reporter either alone or in combination with the ZEB expression vector (kindly provided by D Dean). Luciferase activity was determined in proliferating (GM) or differentiating (DM) myocytes at the indicated times. Error bars represent standard deviations from three independent experiments. (f) ZEB represses the MyoD-dependent activation of the P2p73 promoter. 10T/2 cells were transiently transfected with the P2p73-luc reporter either alone or in combination with the indicated expression vectors (pGal4-MyoD is a gift of Dr PL Puri). (g) In vivo occupancy of the DNp73 promoter. ChiP experiments were previously described (Costanzo et al., 2002). Cross-linked chromatin from proliferating and differentiating (12, 24, and 48 h) C2C12 cells was immunoprecipited with the indicated antibodies (anti-p53 polyclonal Ab-7 from Oncogene Sci; anti-MyoD (M-318) and anti-p73 (C-20 and H-79) polyclonal antibodies from Santa Cruz Biotechnology; antiacetyl-H4 from Upstate Biotech.) and analysed by PCR with specific DNp73 promoter primers (up: 50-CTGAC TGCTCATGCTTTAGAGTGT-30; down: 50-CGCCCCTTTATT CCTTATGACTAT-30).

Article Snippet: Immunoblotting was performed as described (Costanzo et al., 2002) using a pan-p73 mouse monoclonal antibody (Imgenex #1288).

Techniques: Activity Assay, Transfection, Luciferase, Expressing, Plasmid Preparation, Activation Assay, In Vivo

Journal: Oncogene

Article Title: DNp73alpha protects myogenic cells from apoptosis.

doi: 10.1038/sj.onc.1209321

Figure Lengend Snippet: Figure 3 (a) C2C12 cells were transiently transfected using Lipofectamine reagent (Invitrogen) with MLCbgal reporter gene (Sartorelli et al., 1999) either alone or in combination with the indicated plasmids. After 48 h in DM, b-galactoside activity in situ was assayed (Wu et al., 2000). Results are expressed as the percentage of positive blue nuclei. Means from three independent experiments 7s.d. are shown. (b) Left panels: C2C12 cells were cotransfected using the Lipofectamine Plus reagent (Invitrogen) with the indicated siRNA specific for the p73 and p53 family members at a final concentration of 50 nM (sequences available upon request) together with the HA-tagged expression vectors for the indicated proteins. Middle panel: C2C12 cells were transiently cotransfected with the MLCbgal reporter gene either alone or in combination with the indicated specific siRNA and then placed in differentiation medium. b-galactoside activity is analysed as in (a). Right panels: results from three experiments 7s.d. are shown.

Article Snippet: Immunoblotting was performed as described (Costanzo et al., 2002) using a pan-p73 mouse monoclonal antibody (Imgenex #1288).

Techniques: Transfection, Activity Assay, In Situ, Concentration Assay, Expressing

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: Eighty-six genes (brown) were down regulated in the absence of all three p53 family members. A number of genes were down regulated in the absence of each p53 family member individually; 109 in p53 deficient cells (red), 148 in p63 deficient cells (yellow), and 131 in p73 deficient cells (blue). Several genes were down regulated in the absence of two family members; 47 in the absence of p53 and p63 (orange), 58 in the absence of p63 and p73 (green), and 41 in the absence of p53 and p73 (purple).

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques:

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: Each row represents the specified gene. Each column represents the expression level of a specified knock out MEF line relative to the expression level of wild-type MEFs after DNA damage. The red color indicates upregulation, the green color indicates down regulation, while black indicates no significant change of the indicated gene expression. Clustering based on Euclidean distance indicates that p63- and p73- deficient E1A MEFs are more similar to each other than to p53−/− E1A MEFs. Genes of interest are listed in boxes and are associated with their corresponding location on the heatmap.

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques: Expressing, Knock-Out, Gene Expression

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: p53 family response elements assayed by ChIP.

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques:

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: Real time PCR analysis of E1A MEFs of the following genotypes (wild-type, p53−/− , p63−/−, p73−/− and p63−/−;p73−/− ) after treatment with (A) doxorubicin (0.34 µM) for 12 hours or (B) γ radiation (12 hours). The Y-axis shows the fold induction. Bars represent 3 MEF lines for each genotype, each performed in triplicate. Data represent the mean ± SEM. The asterisk denotes statistical significance compared to wild-type, p<0.001.

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques: Real-time Polymerase Chain Reaction

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: (A) Western blot analysis for Rad51 using whole cell lysates from wild-type and p63−/−;p73−/− MEFs treated with 0 Gy or 10 min (m), 30 m, 1 hour (h), 2 h and 4 h after 5 Gy of gamma irradiation. Actin was used as a control for equal loading. (B–I) Immunohistochemistry (IHC) of normal mammary tissue or mammary adenocarcinomas from p63+/−;p73+/− mice using antibodies as follows: (B) normal mammary tissue from p63+/−;p73+/− mouse using Rad51 antibody, (C) mammary adenocarcinoma from p63+/−;p73+/− mouse using Rad51 antibody, (D) normal mammary tissue from p63+/−;p73+/− mouse using BRCA2 antibody, (E) mammary adenocarcinoma from p63+/−;p73+/− mouse using BRCA2 antibody, (F) normal mammary tissue from p63+/−;p73+/− mouse using p63 antibody, (G) mammary adenocarcinoma from p63+/−;p73+/− mouse using p63 antibody, (H) normal mammary tissue from p63+/−;p73+/− mouse using p73 antibody, (I) mammary adenocarcinoma from p63+/−;p73+/− mouse using p73 antibody.

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques: Western Blot, Irradiation, Control, Immunohistochemistry

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: Chromatin immunoprecipitation (ChIP) analysis using wild-type E1A MEFs (WT) and E1A MEFs deficient for the p53 family members ( p53−/− , p63−/− and p73−/− ) before (U) and after treatment with doxorubicin (D) for 12 hours. Antibodies used to immunoprecipitate protein-DNA complexes in each cell line are shown in various colors: p53 (red), p63 (blue), and p73 (green). Total input chromatin is shown for each sample (input). Each ChIP was performed using 3 independent MEF lines in triplicate.

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques: Chromatin Immunoprecipitation

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: Bar graphs showing fold induction for each luciferase reporter gene in (A–D) p63−/−; p73−/− or (E,F) p53−/−;p73−/− primary MEFs. Reporter genes used are as follows: (A,E) pGL3-Rad51-1 containing the binding elements in intron 1, (B) pGL3-Rad51-2 containing the binding elements in intron 2, (C,F) pGL3-BRCA2 containing the binding element in intron 2, and (D) pGL3-mre-11 containing the binding element in intron 1. Pluses above each bar graph indicate which isoforms of p63 or p73 were transfected in cells with the firefly-luciferase reporter genes. Renilla-luciferase was used as a control for transfection efficiency, and pPERP-luc was used as a positive control. Each experiment was performed 6 times using 3 independent MEF lines. Data are represented as the mean ± SEM.

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques: Luciferase, Binding Assay, Transfection, Control, Positive Control

Journal: Oncogene

Article Title: Restoring PUMA induction overcomes KRAS -mediated resistance to anti-EGFR antibodies in colorectal cancer

doi: 10.1038/s41388-018-0289-x

Figure Lengend Snippet: (A) DiFi cells transfected with control scrambled or p73 siRNA for 24 hr were re-plated and treated with 10 nM cetuximab (Cmab). Expression of p73 at 8 hr, and PUMA and cleaved (C) caspase-3 at 24 hr after cetuximab treatment was analyzed by western blotting. Cells without siRNA transfection and re-plating were used as the control for analyzing p73 at 8r after treatment. (B) Western blotting of indicated proteins in DiFi cells treated 10 nM cetuximab at the indicated time points. Phospho-p73 (p-p73, Y99); phospho-AKT (p-AKT, S473); phospho-ERK1/2 (p-ERK1/2, T202/Y204). (C) DiFi cells transfected with either a control empty vector or a HA-p73α construct were treated with 10 nM Cmab for the indicated times. Binding of transfected p73α to the PUMA promoter was analyzed by chromatin immunoprecipitation (ChIP) using anti-HA antibody with IgG as control, followed by PCR amplification and analysis of PCR products by agarose gel electrophoresis. (D) Crystal violet staining (upper panel) and MTS analysis (lower panel) of DiFi cells transfected with siRNA as in (A) and treated with Cmab at the indicated doses for 72 hr. (E) Western blotting of indicated proteins in DiFi cells transfected with control empty vector or constitutively active AKT for 6 hr, and then treated with 10 nM cetuximab for 8 or 24 hr. (F) Crystal violet staining (upper panel) and MTS analysis (lower panel) of DiFi cells transfected as in (E) and treated with Cmab at the indicated doses for 72 hr. (G) A model of PUMA induction by anti-EGFR antibodies.

Article Snippet: Western blotting was performed as previously described [ ] using antibodies against: β-Actin (A5441, Sigma), cleaved caspase-3 (#9661, Cell Signaling, Danvers, MA, USA), cleaved caspase-9 (#9502, Cell Signaling), Mcl-1 (#559027, BD Biosciences, San Jose, CA, USA), Bax (#610983, BD Biosciences), Bid (#2002, Cell Signaling), Bcl-xL (#610212, BD Biosciences), Bim (#2819, Cell Signaling), Bcl-2 (#M0887, Agilent DAKO, Santa Clara, CA, USA), cytochrome c (sc-7159, Santa Cruz Biotechnology, Santa Cruz, CA, USA), COX IV (A21348, Invitrogen), PUMA [ ], Bak (#06–536, EMD Millipore), Noxa (#OP180, EMD Millipore), p73 (A300–126A, Bethyl Laboratories, Montgomery, TX, USA), p-p73 (#4665, Cell Signaling), p-AKT (#4058, Cell Signaling), total AKT (#9272, Cell Signaling), p-ERK1/2 (#4376, Cell Signaling), total ERK1/2 (#9102, Cell Signaling), p-FoxO3A (#9464, Cell Signaling), total FoxO3A (07–702, EMD Millipore), p53 (sc-126, Santa Cruz), p-EGFR (#2234, Cell Signaling), total EGFR (#610016, BD Biosciences), KRAS (sc-30, Santa Cruz), p-Aurora A/B/C (#2914, Cell Signaling), total Aurora A (#4718, Cell Signaling), total Aurora B (#3094, Cell Signaling), and HA (#12CA5, Roche, Indianapolis, IN, USA).

Techniques: Transfection, Control, Expressing, Western Blot, Plasmid Preparation, Construct, Binding Assay, Chromatin Immunoprecipitation, Amplification, Agarose Gel Electrophoresis, Staining

Journal: Oncogene

Article Title: Restoring PUMA induction overcomes KRAS -mediated resistance to anti-EGFR antibodies in colorectal cancer

doi: 10.1038/s41388-018-0289-x

Figure Lengend Snippet: (A) MTS analysis of parental (red) and cetuximab-resistant (Cmab-R, black) DiFi cells treated with cetuximab (Cmab) or panitumumab (Pmab) at the indicated doses for 72 hr. (B) Western blotting of cleaved (C) caspase-3 in the parental and Cmab-R DiFi cells treated with 10 nM of Cmab or Pmab for 24 hr. (C) Western blotting of indicated proteins in the parental and Cmab-R DiFi cells. Lysates of Cmab-R DiFi cells were prepared from cells cultured in medium with 10 nM cetuximab (Cmab+), or without cetuximab (Cmab-) for 6 days. p-EGFR (Y1068); p-AKT (S473); p-ERK1/2 (T202/Y204). (D) Western blotting of indicated Bcl-2 family proteins in the parental and Cmab-R DiFi cells treated with 10 nM of Cmab for 24 hr. (E) Western blotting of phosphorylated (p-p73, Y99) and total p73 in the parental and Cmab-R DiFi cells treated with 10 nM Cmab for 8 hr. (F) Parental and Cmab-R DiFi cells transfected with control empty or HA-p73α-expressing vector were treated with 10 nM cetuximab for 8 hr. Binding of transfected p73α to the PUMA promoter was analyzed by chromatin immunoprecipitation (ChIP) using anti-HA antibody, followed by PCR amplification and analysis of PCR products by agarose gel electrophoresis. (G) Parental and Cmab-R DiFi cells were infected with EGFP-PUMA-expressing adenovirus (Ad-PUMA) at the indicated MOI for 24 hr. Upper, analysis of apoptosis by nuclear fragmentation; lower , western blotting of PUMA. Results were expressed as means ± s.e.m. of triplicates in two independent experiments. *** P <0.001.

Article Snippet: Western blotting was performed as previously described [ ] using antibodies against: β-Actin (A5441, Sigma), cleaved caspase-3 (#9661, Cell Signaling, Danvers, MA, USA), cleaved caspase-9 (#9502, Cell Signaling), Mcl-1 (#559027, BD Biosciences, San Jose, CA, USA), Bax (#610983, BD Biosciences), Bid (#2002, Cell Signaling), Bcl-xL (#610212, BD Biosciences), Bim (#2819, Cell Signaling), Bcl-2 (#M0887, Agilent DAKO, Santa Clara, CA, USA), cytochrome c (sc-7159, Santa Cruz Biotechnology, Santa Cruz, CA, USA), COX IV (A21348, Invitrogen), PUMA [ ], Bak (#06–536, EMD Millipore), Noxa (#OP180, EMD Millipore), p73 (A300–126A, Bethyl Laboratories, Montgomery, TX, USA), p-p73 (#4665, Cell Signaling), p-AKT (#4058, Cell Signaling), total AKT (#9272, Cell Signaling), p-ERK1/2 (#4376, Cell Signaling), total ERK1/2 (#9102, Cell Signaling), p-FoxO3A (#9464, Cell Signaling), total FoxO3A (07–702, EMD Millipore), p53 (sc-126, Santa Cruz), p-EGFR (#2234, Cell Signaling), total EGFR (#610016, BD Biosciences), KRAS (sc-30, Santa Cruz), p-Aurora A/B/C (#2914, Cell Signaling), total Aurora A (#4718, Cell Signaling), total Aurora B (#3094, Cell Signaling), and HA (#12CA5, Roche, Indianapolis, IN, USA).

Techniques: Western Blot, Cell Culture, Transfection, Control, Expressing, Plasmid Preparation, Binding Assay, Chromatin Immunoprecipitation, Amplification, Agarose Gel Electrophoresis, Infection

Journal: American Journal of Physiology - Cell Physiology

Article Title: Protein kinase G increases antioxidant function in lung microvascular endothelial cells by inhibiting the c-Abl tyrosine kinase

doi: 10.1152/ajpcell.00375.2012

Figure Lengend Snippet: A: wild-type mouse lung microvascular endothelial cells (MLMVEC). Representative Western blot showing the effect of 0, 250, and 500 μM H2O2 on phospho-Y245 c-Abl and total c-Abl in the presence and absence of 8pCPT-cGMP. Graph shows effect of 30-min 8pCPT-cGMP (50 μM) on H2O2-induced c-Abl activation in wild-type MLMVEC, as measured by c-Abl Y245 phosphorylation, controlled for total c-Abl (n = 5; *P < 0.05, significant interaction by ANOVA). B: wild-type MLMVEC. Representative Western blot showing the effect of 0, 250, and 500 μM H2O2 on phospho-Y99 p73 and total p73 in the presence and absence of 8pCPT-cGMP. Graph shows effect of 30-min 8pCPT-cGMP (50 μM) on H2O2-induced p73 phosphorylation in wild-type MLMVEC, controlled for total p73 (n = 6; *P < 0.05 by ANOVA). C: protein kinase GI mice (PKGI−/−) MLMVEC. Representative Western blots showing phosphorylation of vasodilator-stimulated phosphoprotein (VASP) serine 235 after 8pCPT-cGMP treatment in wild-type but not PKGI−/− MLMVEC, and the effect of 0, 250, and 500 μM H2O2 on phospho-Y245 c-Abl and total c-Abl in the presence and absence of 8pCPT-cGMP. D, left: graph shows effect of 30-min 8pCPT-cGMP on H2O2-induced c-Abl activation in PKGI−/− MLMVEC (n = 4; P = not significant). Data are normalized to wild-type mean. D, right: graph shows baseline c-Abl activity, as measured by phospho-Y245 c-Abl/total c-Abl ratio in wild-type vs. PKGI−/− MLMVEC in the absence of 8pCPT-cGMP or H2O2 exposure (n = 9 and 4; *P < 0.01). All values are presented as means ± SE.

Article Snippet: Total p73 and total c-Abl antibodies were from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Western Blot, Activation Assay, Activity Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Protein kinase G increases antioxidant function in lung microvascular endothelial cells by inhibiting the c-Abl tyrosine kinase

doi: 10.1152/ajpcell.00375.2012

Figure Lengend Snippet: A: wild-type MLMVEC. Representative western blot showing the effect of 0, 250, and 500 μM H2O2 on phospho-Y245 c-Abl and total c-Abl in the presence and absence of atrial natriuretic peptide (ANP; 10 nM). Graph shows effect of 30-min ANP (10 nM) on H2O2-induced c-Abl activation in wild-type MLMVEC, as measured by c-Abl Y245 phosphorylation, controlled for total c-Abl (n = 7; *P < 0.05 by ANOVA). B: wild-type MLMVEC. Representative Western blots showing the effect of 0, 250, and 500 μM H2O2 on phospho-Y99 p73 and total p73 in the presence and absence of ANP. Graph shows effect of H2O2 on p73 phosphorylation, controlled for total p73 (n = 7; *P < 0.05 for effect of H2O2 in diluent-treated cells by ANOVA). The absence of phospho-p73 in ANP-treated cells (30 min, 10 nM) precluded quantification; signal represented as zero in graph.

Article Snippet: Total p73 and total c-Abl antibodies were from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Western Blot, Activation Assay