Journal: Nucleic Acids Research

Article Title: PHD finger protein 1 (PHF1) is a novel reader for histone H4R3 symmetric dimethylation and coordinates with PRMT5–WDR77/CRL4B complex to promote tumorigenesis

doi: 10.1093/nar/gky461

Figure Lengend Snippet: PHF1 Interacts with CRL4B and PRMT5–WDR77 Complex. ( A ) Immunoaffinity purification of PHF1-containing protein complexes. Cellular extracts from HEK 293T cells stably expressing FLAG (control) or FLAG-PHF1 were immunopurified with anti-FLAG affinity columns and eluents with FLAG peptide. The eluates were resolved by SDS-PAGE and silver-stained. The protein bands were retrieved and analyzed by mass spectrometry. Detailed results from the mass spectrometric analysis are provided in . ( B ) Western blot analysis of the purified fractions using antibodies against the indicated proteins. ( C ) Association of PHF1 with the P5-W77 (PRMT5, WDR77) complex, the CRL4B complex (CUL4B, DDB1, ROC1), the NuRD components (MTA1, MTA3, LSD1, HDAC1, HDAC2), the SIN3A complex (SIN3A, SAP180, SAP30) and the p160 SRC family (SRC-1, GRIP-1, AIB-1) in HEK 293T cells. Whole cell lysates from HEK 293T cells were prepared and co-immunoprecipitation (co-IP) assays with anti-PHF1 followed by immunoblotting (IB) with antibodies against the indicated proteins, or with antibodies against the indicated proteins followed by IB with anti-PHF1. PHF1 did not interact with the PRC1 complex (BMI1, RING1A, RING1B). IgG served as a negative control. This figure consists of seven sub-panels which showed the association of PHF1 with the seven indicated complexes. In the left part of each sub-panel, co-IP assays were performed with PHF1 antibody; immunocomplexes were then immunoblotted using antibodies against the intermediate proteins. In the right part of each sub-panel, co-IP assays were performed with antibodies against each of intermediate proteins respectively and then immunoblotted using PHF1’s antibody. The slanting labels identify the IP antibodies, and the horizontal labels in the middle indicate the detailed IB antibodies for the left panels and detailed IP antibodies for the right panels. ( D ) Association of CRL4B complex with PRMT5–WDR77 complex in HEK 293T cells. Whole cell lysates from HEK 293T cells were prepared and co-IP was performed with antibodies against the indicated proteins. Immunocomplexes were then IB tested using antibodies against the indicated proteins. The figure consists of two sub-panels which showed the association of PRMT5–WDR77 with CRL4B complex. In the left part of each sub-panel, co-IP assays were performed with PRMT5 antibody in the upper sub-panel and WDR77 antibody in the lower sub-panel; immunocomplexes were then immunoblotted using antibodies against the intermediate proteins. In the right part of each sub-panel, co-IP assays were performed with antibodies against each of intermediate proteins respectively and then immunoblotted using PRMT5’s antibody in the upper sub-panel and WDR77’s antibody in the lower sub-panel. The slanting labels identify the IP antibodies, and the horizontal labels of CRL4B complex in the middle indicate the detailed IB antibodies for the left panels and detailed IP antibodies for the right panels. ( E ) Association of PHF1, PRMT5–WDR77 complex, and CRL4B complex in MDA-MB-231 and Hs 578T cells. Whole cell lysates were immunoprecipitated with antibodies against the indicated proteins. Immunocomplexes were then IB tested using antibodies against the indicated proteins. ( F ) Co-fractionation of PHF1, PRMT5–WDR77 complex, and CRL4B complex by fast protein liquid chromatography (FPLC). Nuclear extracts of MDA-MB-231 cells were fractionated on a DEAE sepharose column followed by a Superose 6 gel filtration column. The fractions were analyzed by western blotting. Molecular weight standards are shown at top (in kDa). ( G ) Western blot of the PHF1-containing complex fractionated by Superose 6 gel filtration.

Article Snippet: Next, we determined the expression profile of PHF1 using the human cancer survey RT-qPCR gene expression panel (Origene) in tumors originating from breast, colon, kidney, lung, ovary, and thyroid tissue (Figure ).

Techniques: Immunoaffinity Purification, Stable Transfection, Expressing, SDS Page, Staining, Mass Spectrometry, Western Blot, Purification, Immunoprecipitation, Co-Immunoprecipitation Assay, Negative Control, Fractionation, Fast Protein Liquid Chromatography, Filtration, Molecular Weight

Journal: Nucleic Acids Research

Article Title: PHD finger protein 1 (PHF1) is a novel reader for histone H4R3 symmetric dimethylation and coordinates with PRMT5–WDR77/CRL4B complex to promote tumorigenesis

doi: 10.1093/nar/gky461

Figure Lengend Snippet: Molecular Interaction between PHF1, PRMT5–WDR77, and CRL4B Complex subunits. ( A, B ) GST pull-down experiments with bacterially expressed GST-fused proteins and the indicated in vitro transcribed/translated proteins. The slanting labels show the GST-fused proteins, the vertical labels indicate the IB antibodies, and the horizontal labels in the right panels show the detailed GST-fused proteins. ( C, D ) Identification of the essential domains required for the interaction with PRMT5 or DDB1 of PHF1. GST pull-down experiments with a bacterially expressed series of truncation constructs of PHF1 (N1–N5, C1–C5) to generate GST fusion proteins and the in vitro transcribed/translated indicated proteins. ( E, F ) Identification of the essential domains required for interactions with PHF1 or WDR77 of DDB1. GST pull-down experiments with bacterially expressed GST-fused truncated DDB1 proteins and the indicated in vitro transcribed/translated proteins. ( G ) Schematic diagram depicting the molecular interactions between PHF1, PRMT5–WDR77 and CRL4B.

Article Snippet: Next, we determined the expression profile of PHF1 using the human cancer survey RT-qPCR gene expression panel (Origene) in tumors originating from breast, colon, kidney, lung, ovary, and thyroid tissue (Figure ).

Techniques: In Vitro, Construct

Journal: Nucleic Acids Research

Article Title: PHD finger protein 1 (PHF1) is a novel reader for histone H4R3 symmetric dimethylation and coordinates with PRMT5–WDR77/CRL4B complex to promote tumorigenesis

doi: 10.1093/nar/gky461

Figure Lengend Snippet: The PHD domain of PHF1 recognizes H4R3me2s. ( A ) GST pull-down experiments with bacterially expressed GST-PHF1 and histone octamers. Immunoblot using antibodies against various histone methylation modifications. ( B ) Whole cell lysates from HEK 293T cells were immunoprecipitated with antibodies against PHF1. Immunocomplexes were then IB tested using antibodies against the indicated proteins. ( C ) GST pull-down experiments with bacterially expressed GST-PHF1 and peptides with indicated residues. ( D ) GST pull-down experiments with bacterially expressed GST-fused truncated PHF1 proteins and peptides with H4R3me2s or H3K36me3. ( E ) Anti-His immunoblot (left panel) and graphical analysis of the highest binding events detected (right panel) showing the binding specificity of the His-PHF1 PHD1 domain measured on Histone Peptide Array. Peptides were spotted in duplicate as shown in two boxes on the same array. The positions of H4R3me2s-containing peptides are highlighted with blue circles and H4R3me2a with red circles. ( F ) Scatter plot of duplicate peptide arrays probed with the PHF1 PHD1 domain. Peptides are colored according to the legend. The correlation coefficient was calculated to measure linear association. ( G ) Graphical representation of H4R3me2-containing peptides showing the binding specificity of GST-PHF1 PHD1 measured on Histone Peptide Array. ( H ) Experimental ITC titration curves of PHF1 PHD1 binding to differentially methylated H4R3 peptides, as indicated. Concentrations of protein and peptide were approximately 50 and 500 μM, respectively. The parameters are labeled beside the figures. N.D., not determined. ( I ) SPR analysis of the interaction of PHF1 PHD1 with differentially methylated H4R3 peptides, as indicated. The parameters are labeled in the figures. N.D., not determined. ( J ) Whole cell lysates from HEK 293T cells transfected with indicated FLAG-PHD1 mutations were immunoprecipitated with antibodies against FLAG. Immunocomplexes were then IB tested using antibodies against H4R3me2s. ( K ) GST pull-down experiments with the indicated bacterially expressed GST-fused PHD1 mutations and H4R3me2s peptides. ( L ) Western blot analysis of chromatin-bound and soluble cell fractions in HEK 293T cells after transfection with control shRNA or shRNA targeting PRMT5. Gray scanning analysis of the PHF1 blot is shown in the bottom panel. ( M ) Western blot analysis of chromatin-bound and soluble cell fractions in HEK 293T cells after transfection with control shRNA or shRNA targeting SETD2.

Article Snippet: Next, we determined the expression profile of PHF1 using the human cancer survey RT-qPCR gene expression panel (Origene) in tumors originating from breast, colon, kidney, lung, ovary, and thyroid tissue (Figure ).

Techniques: Western Blot, Methylation, Immunoprecipitation, Binding Assay, Peptide Microarray, Titration, Labeling, Transfection, shRNA

Journal: Nucleic Acids Research

Article Title: PHD finger protein 1 (PHF1) is a novel reader for histone H4R3 symmetric dimethylation and coordinates with PRMT5–WDR77/CRL4B complex to promote tumorigenesis

doi: 10.1093/nar/gky461

Figure Lengend Snippet: Genome-wide Transcriptional Target Analysis for the PHF1/PRMT5/CRL4B Complex. ( A ) Pathway analysis of the 703 target genes of PHF1 arranged into functional groups. ( B ) Venn diagram of overlapping promoters bound by PHF1, PRMT5 and CUL4B in MDA-MB-231 cells. The numbers represent the number of the promoters that were targeted by the indicated proteins. The detailed results of the ChIP-on-chip experiments are provided in . ( C – E ) Verification of the ChIP-on-chip results by qChIP analysis of the indicated genes in MDA-MB-231 cells. Results are presented as percentage of input with glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) as a negative control. Error bars represent mean ± SD for three independent experiments ( ∗ P < 0.05, ∗∗ P < 0.01, and two-tailed unpaired t test).

Article Snippet: Next, we determined the expression profile of PHF1 using the human cancer survey RT-qPCR gene expression panel (Origene) in tumors originating from breast, colon, kidney, lung, ovary, and thyroid tissue (Figure ).

Techniques: Genome Wide, Functional Assay, Negative Control, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: PHD finger protein 1 (PHF1) is a novel reader for histone H4R3 symmetric dimethylation and coordinates with PRMT5–WDR77/CRL4B complex to promote tumorigenesis

doi: 10.1093/nar/gky461

Figure Lengend Snippet: Tumor suppressor genes E-cadherin and FBXW7 are cotargeted by PHF1/PRMT5/CRL4B complex. ( A ) The efficiency of four shRNAs targeting PHF1, PRMT5 or CUL4B , respectively. MDA-MB-231 cells were infected with lentiviruses carrying control shRNA (shSCR) or four different shRNAs targeting PHF1, PRMT5 or CUL4B . The knockdown efficiencies of PHF1, PRMT5 , and CUL4B were verified by RT-qPCR. We chose shPHF1#2, shPRMT5#3, and shCUL4B#2 (marked in red) for further study. Error bars represent mean ± SD for three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (two-tailed unpaired t test). ( B and C ) Clones in which PHF1, PRMT5 , or CUL4B were stably knocked down were compared with the parental cell lines with respect to the levels of mRNA and protein of E-cadherin and FBXW7 in MDA-MB-231, Hs 578T and MDA-MB-453 cells. The mRNA levels were normalized to those of GAPDH and β-actin served as a loading control for western blotting. Error bars represent mean ± SD of three independent experiments. ( ∗ P < 0.05, ∗∗ P < 0.01, and two-tailed unpaired t test). ( D ) PHF1, PRMT5–WDR77, and CRL4B complexes exist in the same protein complex on the E-cadherin and FBXW7 promoters. CUL4A served as a negative control. ChIP and Re-ChIP experiments were performed in MDA-MB-231 cells with the indicated antibodies. ( E ) qChIP analysis of the recruitment of the indicated proteins on E-cadherin and FBXW7 promoters in MDA-MB-231 cells after transfection with control shRNA or shRNAs targeting PHF1, PRMT5 or CUL4B. Purified rabbit IgG was used as a negative control. Error bars represent mean ± SD of three independent experiments ( ∗ P < 0.05, ∗∗ P < 0.01, and two-tailed unpaired t test).

Article Snippet: Next, we determined the expression profile of PHF1 using the human cancer survey RT-qPCR gene expression panel (Origene) in tumors originating from breast, colon, kidney, lung, ovary, and thyroid tissue (Figure ).

Techniques: Infection, shRNA, Quantitative RT-PCR, Two Tailed Test, Clone Assay, Stable Transfection, Western Blot, Negative Control, Transfection, Purification

Journal: Nucleic Acids Research

Article Title: PHD finger protein 1 (PHF1) is a novel reader for histone H4R3 symmetric dimethylation and coordinates with PRMT5–WDR77/CRL4B complex to promote tumorigenesis

doi: 10.1093/nar/gky461

Figure Lengend Snippet: PHF1 promotes carcinogenesis and metastasis of breast cancer in vitro and in vivo . ( A ) PHF1 promotes cellular proliferation. Growth curve analysis was performed on MDA-MB-231, Hs 578T, and MDA-MB-453 cells transfected with vector or FLAG-PHF1, or transfected with shSCR, two different PHF1 shRNAs or together with shRNA-resistant FLAG-PHF1 wild type (FLAG-PHF1 res) or H115A, C136A mutants. ( B ) PHF1 enhances the colony-forming efficiency of breast cancer cells. MDA-MB-231, Hs 578T and MDA-MB-453 cells transfected with vector or FLAG-PHF1 were maintained for 4 days while cells transfected with shSCR, two different shRNAs of PHF1 or together with shRNA-resistant FLAG-PHF1 (FLAG-PHF1 res) were maintained in culture media for 7 days prior to crystal violet staining. Representative photos and statistical analyses are shown. ( C ) Western blot analysis of PHF1 expression in MDA-MB-231 cells transfected with different lentiviruses. β-actin served as a loading control. ( D ) MDA-MB-231 cells and Hs 578T cells were incubated with EdU for 2 h. A fluorescence microscope was used for EdU detection. ( E and F ) Expression of the indicated epithelial or mesenchymal markers was measured by RT-qPCR or western blotting in MDA-MB-231, Hs 578T and MDA-MB-453 cells with PHF1 depleted. ( G ) MCF-7, MDA-MB-231, and Hs 578T cells were transfected with vector or FLAG-PHF1 for cell invasion assays using Matrigel transwell filters. MDA-MB-231 cells and Hs 578T cells transfected with shSCR, two different shRNAs of PHF1 or/and siE-cadherin were used for the same assays. The invading cells were stained and counted. The images represent one field under microscopy in each group. ( H and I ) MDA-MB-231-Luc-D3H2LN cells infected with lentiviruses carrying shSCR or shRNA against PHF1 were inoculated orthotopically into the abdominal mammary fat pad of 6-week-old female SCID mice ( n = 6). Primary tumors were quantified using bioluminescence imaging 6 weeks after initial implantation. Representative in vivo bioluminescent images are shown (H), and tumor specimens were examined by in vitro bioluminescent measurement (I). ( J and K ) The above-described MDA-MB-231-Luc-D3H2LN cells were injected intravenously through the tail vein of 6-week-old female SCID mice ( n = 6). Lung metastases were quantified using bioluminescence imaging after 6 weeks. Representative in vivo bioluminescent images are shown (J). Lung cancer specimens were examined by in vitro bioluminescent measurement (K). (A–K) Two-tailed unpaired t test ( ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001).

Article Snippet: Next, we determined the expression profile of PHF1 using the human cancer survey RT-qPCR gene expression panel (Origene) in tumors originating from breast, colon, kidney, lung, ovary, and thyroid tissue (Figure ).

Techniques: In Vitro, In Vivo, Transfection, Plasmid Preparation, shRNA, Staining, Western Blot, Expressing, Incubation, Fluorescence, Microscopy, Quantitative RT-PCR, Infection, Imaging, Injection, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: PHD finger protein 1 (PHF1) is a novel reader for histone H4R3 symmetric dimethylation and coordinates with PRMT5–WDR77/CRL4B complex to promote tumorigenesis

doi: 10.1093/nar/gky461

Figure Lengend Snippet: Expression of PHF1 is upregulated in breast cancer and negatively correlated with expression of E-cadherin and FBXW7. ( A ) Immunohistochemical staining of PHF1 in normal breast tissues and breast carcinomas (histological grades I, II, and III). ( B ) Positively stained nuclei (in percentages) in grouped samples were analyzed by two-tailed unpaired t test ( ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001). ( C and D ) Analysis of public datasets (GSE43379, GSE32641 and GSE54140) for the expression of PHF1 by two-tailed unpaired t test ( ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001). ( E ) Analysis of public datasets (GSE9195, GSE35428, and GSE36774) for the expression of PHF1 and E-cadherin/FBXW7 in breast cancer. The relative levels of E-cadherin or FBXW7 were plotted against that of PHF1. ( F ) Kaplan–Meier survival analysis for the relationship between survival time and PHF1, PRMT5, WDR77, CUL4B, DDB1 and E-cadherin signature in breast cancer using the online tool ( http://kmplot.com/analysis/ ).

Article Snippet: Next, we determined the expression profile of PHF1 using the human cancer survey RT-qPCR gene expression panel (Origene) in tumors originating from breast, colon, kidney, lung, ovary, and thyroid tissue (Figure ).

Techniques: Expressing, Immunohistochemical staining, Staining, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: PHD finger protein 1 (PHF1) is a novel reader for histone H4R3 symmetric dimethylation and coordinates with PRMT5–WDR77/CRL4B complex to promote tumorigenesis

doi: 10.1093/nar/gky461

Figure Lengend Snippet: Expression of PHF1 is upregulated in multiple carcinomas and is a potential cancer biomarker. ( A and B ) PHF1 is upregulated in multiple carcinomas. Immunohistochemical staining of PHF1 in paired samples of breast, lung, colon, stomach, ovary, prostate, esophageal, liver, pancreas, kidney, rectal and uterine carcinoma versus adjacent normal tissues. Representative images of 200-fold magnifications of each type of paired tumor section are presented. Each bar represents the mean ± SD for triplicate experiments ( ∗ P < 0.05 and ∗∗ P < 0.01). ( C ) Transcript levels of PHF1 are significantly higher in multiple malignant tumor tissues. Relative levels of PHF1 transcripts were normalized to that of GAPDH and calibrated to the mean mRNA level (arbitrary value of 1) in normal tissue (yellow bars). The fold increase in gene expression relative to the mean value for each disease sample is indicated. ( D ) PHF1 expression in multiple cancer microarray datasets available from Oncomine ( https://www.oncomine.com/ ). ( E ) Analysis of public datasets (GSE19804, GSE64258, GSE50830, GSE66229, GSE17951, GSE19632, GSE28735, GSE17025 and GSE25638) for the expression of PHF1 and E-cadherin in multiple carcinomas. The relative level of E-cadherin was plotted against that of PHF1 . ( F ) Kaplan-Meier survival analysis for the relationship between survival time and PHF1 signature in gastric cancer, lung cancer and ovarian cancer using the online tool. ( G ) Graphic model as discussed in the text. DNA (black line); nucleosomes with single N-terminus of H4 and C-terminus of H2A (blue ball). (a) PHF1 recognized the H4R3me2s modification. (b) PRMT5/WDR77 and CRL4B were recruited by PHF1, and the PHF1/PRMT5–WDR77/CRL4B complex was formed on the target promoters, which was stable and further catalyzed H4R3me2s/H2AK119ub1 formation to establish, as well as maintain repression at these sites.

Article Snippet: Next, we determined the expression profile of PHF1 using the human cancer survey RT-qPCR gene expression panel (Origene) in tumors originating from breast, colon, kidney, lung, ovary, and thyroid tissue (Figure ).

Techniques: Expressing, Biomarker Assay, Immunohistochemical staining, Staining, Microarray, Modification

Journal: The Journal of Biological Chemistry

Article Title: Polycomb Group Protein PHF1 Regulates p53-dependent Cell Growth Arrest and Apoptosis *

doi: 10.1074/jbc.M111.338996

Figure Lengend Snippet: PHF1 specifically activates p53 pathway. A, HepG2 cells were transfected with the indicated luciferase pathway reporter construct (100 ng), pTK-galactosidase (20 ng) construct and PHF1 expression construct (150 ng) or with the empty vector (150 ng). At 24 h after transfection, the luciferase activities were measured by luminometer. B, H1299, U2OS, HCT116 p53+/+, and HCT116 p53−/− cells were transfected, respectively, with the p53 responsive element reporter (p53-Luc, 100 ng) along with pTK-galactosidase (20 ng) and an increasing amount of PHF1 (100, 200 ng) and empty construct as indicated. At 24 h after transfection, the luciferase activities were measured by luminometer. C, HepG2 and U2OS cells were transfected with the p21 and Bax promoter reporters (p21-Luc/Bax-Luc, 100 ng), pTK-galactosidase (20 ng), and increasing amount of PHF1 (100 or 200 ng) or empty constructs as indicated. At 24 h after transfection, the luciferase activities were measured by luminometer. The mean value (± S.D.) of three independent experiments is shown.

Article Snippet: RNA Interference The HuSH 29-mer shRNA constructs against PHF1 in pGFP-V-RS vector were purchased from OriGene, Inc.

Techniques: Transfection, Luciferase, Construct, Expressing, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Polycomb Group Protein PHF1 Regulates p53-dependent Cell Growth Arrest and Apoptosis *

doi: 10.1074/jbc.M111.338996

Figure Lengend Snippet: PHF1 interacts with p53 both in vivo and in vitro. A, H1299 cells were transfected with Myc-PHF1 and/or HA-p53 as indicated. Cell lysates were prepared and subjected to immunoprecipitation (IP) with anti-HA or Myc antibody. The immunoprecipitates were detected by Western blot (WB) with anti-Myc and anti-HA antibodies, respectively. B, co-immunoprecipitation of endogenous PHF1 and p53 proteins in U2OS cells. The endogenous p53 was immunoprecipitated by p53 (FL-393) antibody with rabbit IgG as a negative control. The immunoprecipitates were detected by Western blot with anti-PHF1 and p53 antibodies, respectively. C, H1299 cells were transfected with Myc-p53 and/or (&.) GFP-PHF1. Cells were fixed 48 h after transfection and subjected to immunofluorescence analysis. D, bacterially expressed GST fusion PHF1 protein bound to glutathione-Sepharose beads and incubated with His-tagged fusion p53 protein. Bound His-p53 was detected by Western blot with anti-His antibody. M.W., molecular weight.

Article Snippet: RNA Interference The HuSH 29-mer shRNA constructs against PHF1 in pGFP-V-RS vector were purchased from OriGene, Inc.

Techniques: In Vivo, In Vitro, Transfection, Immunoprecipitation, Western Blot, Negative Control, Immunofluorescence, Incubation, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: Polycomb Group Protein PHF1 Regulates p53-dependent Cell Growth Arrest and Apoptosis *

doi: 10.1074/jbc.M111.338996

Figure Lengend Snippet: Delineation of the domains mediating the mutual interactions between PHF1 and p53. A, schematic presentation of p53 domains (left panel, top) and deletion mutants (left panel, bottom) and the results of GST pulldown experiments (right panel). Bacterially expressed GST fusion proteins of wild-type p53 and the deletion mutants were bound to glutathione-Sepharose beads, respectively, and incubated with lysates of H1299 cells transfected with a Myc-PHF1 expression construct. Bound Myc-PHF1 was detected by Western blot with anti-HA antibody, and various GST-p53 proteins were detected by ponceau S staining. B and C, schematic model of PHF1 domains (left panel, top) and deletion mutants (left panel, bottom) and the results of GST pulldown experiments (right panel). Bacterially expressed GST fusion proteins of wild-type PHF1 and the deletion mutants were bound to glutathione-Sepharose beads, respectively, and incubated with lysates of H1299 cells transfected with a Myc-p53 expression construct. Bound Myc-p53 was detected by Western blot with Do-1 antibody, and GST-PHF1 deletion mutant proteins were detected by ponceau S staining. M.W., molecular weight.

Article Snippet: RNA Interference The HuSH 29-mer shRNA constructs against PHF1 in pGFP-V-RS vector were purchased from OriGene, Inc.

Techniques: Incubation, Transfection, Expressing, Construct, Western Blot, Staining, Mutagenesis, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: Polycomb Group Protein PHF1 Regulates p53-dependent Cell Growth Arrest and Apoptosis *

doi: 10.1074/jbc.M111.338996

Figure Lengend Snippet: PHF1 regulates p53 protein stability. A, the increasing amount of WT or various mutant expression constructs of Myc-PHF1 were co-transfected with same amounts of HA-p53 into H1299 cells. The protein levels of PHF1 and p53 were determined by Western blot. The pGFP-N3 expression construct was included as a transfection efficiency control. The quantification of immunoblot is shown in the lower panel. The mean value (± S.D.) of three independent experiments is shown. B, H1299 cells were co-transfected with HA-p53, along with empty vector, or Myc-PHF1 constructs. At 48 h after transfection, cells were treated with 30 μm cycloheximide (CHX) for the indicated lengths of time. Equal amounts of cell lysates were detected by Western blot, and for each experimental condition, the blot corresponds to one representative experiment, and the graph in the lower panel shows the quantification of p53 protein levels using GFP for standardization. The mean values (± S.D.) of three independent experiments are shown. C, U2OS cells and HepG2 cells were transiently transfected with two HuSH 29-mer shRNA constructs (KD-1 and KD-2) against PHF1 or control constructs (NC), and at 72 h after transfection, the protein levels of endogenous PHF1 and p53 were detected by Western blot. The quantification of immunoblots is shown in the right panel. The mean values (± S.D.) of three independent experiments are shown. D, qRT-PCR measurements of the mRNA levels of PHF1 and p53 in PHF1 knockdown U2OS cells. The left three columns are relative mRNA levels of PHF1, and the right three columns correspond to those of p53. The mRNA level of GAPDH was used for normalization. E, U2OS cells were transiently transfected with shRNA constructs against PHF1 (KD-1) or control constructs (NC), at 48 h after transfection, cells were treated with 30 μm cycloheximide (CHX) for the indicated lengths of time. Equal amounts of cell lysates were detected by Western blot. Right panel shows the quantification of p53 protein levels. F, qRT-PCR measurements of the expression of p53 target genes in control and PHF1 knockdown U2OS cells. The mean values (± S.D.) of three independent experiments are shown. G, U2OS cells transiently transfected with PHF1-specific shRNA (KD-1) or control shRNA (NC) were treated with 20 μm etoposide for the indicated time. Cell lysates were detected by Western blot with indicated antibodies.

Article Snippet: RNA Interference The HuSH 29-mer shRNA constructs against PHF1 in pGFP-V-RS vector were purchased from OriGene, Inc.

Techniques: Mutagenesis, Expressing, Construct, Transfection, Western Blot, Plasmid Preparation, shRNA, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Polycomb Group Protein PHF1 Regulates p53-dependent Cell Growth Arrest and Apoptosis *

doi: 10.1074/jbc.M111.338996

Figure Lengend Snippet: PHF1 stabilizes p53 by protecting from MDM2-mediated ubiquitination and degradation. A, H1299 cells were transfected with Myc-PHF1 and/or FLAG-tagged ubiquitin ligases (MDM2, Pirh2, and COP1) and deubiquitinases (USP7 and USP10) as indicated. Cell lysates were prepared and subjected to immunoprecipitation with anti-FLAG antibody. The immunoprecipitates were eluted using 3× FLAG and detected by Western blot with indicated antibodies. B, bacterially expressed and purified GST-PHF1 or GST-PHF1-ΔBD1/2 were incubated with GST-p53 or GST-MDM2 in the presence of E1, E2 (UbcH5a), and ubiquitin. Following the ubiquitination reaction, the p53-ubiquitin conjugates were detected by Western blot with p53 antibody. C, HA-MDM2, pcDNA3.0-p53, Myc-PHF1, and FLAG-ubiquitin were co-transfected into H1299 cells. Cells were treated with of the proteasome inhibitor MG132 (20 μm) for 4 h before harvest. The polyubiquitinated forms of p53 were detected by Western blot with anti-p53 antibody. D, shRNA construct (KD-1) against PHF1 or control construct (NC) were co-transfected into U2OS cells. 48 h after transfection, the cells were treated with 20 μm etoposide for 8 h, the polyubiquitinated forms of endogenous p53 were detected by Western blot with anti-p53 antibody. Equal amount of p53 proteins in both control and PHF1 knockdown groups were loaded. E, HA-p53, FLAG-SUMO1, and Myc-PHF1 were co-transfected into H1299 cells. p53 was immunoprecipitated by anti-HA antibody. The sumoylated forms of p53 were detected by Western blot with anti-p53 antibody. F, the increasing amount of WT or ΔBD1/2 Myc-PHF1 expression constructs were co-transfected with HA-MDM2 and untagged p53 constructs into H1299 cells. The protein levels of PHF1, MDM2, and p53 were determined by Western blot. G, HA-MDM2, pcDNA3.0-p53, Myc-PHF1, or PHF1-ΔBD1/2 mutants were co-transfected into H1299 cells. 36 h after transfection, cells were lysed and subjected to immunoprecipitation using anti-p53 antibody. Immunoprecipitated proteins were detected with the indicated antibodies. H, HA-MDM2, FLAG-ubiquitin, and Myc-PHF1 constructs were co-transfected into H1299 cells. Cells were treated with MG132 for 4 h before harvest. MDM2 was immunoprecipitated by anti-HA antibody. The polyubiquitinated forms of MDM2 were detected by Western blot with anti-FLAG antibody. M.W., molecular weight.

Article Snippet: RNA Interference The HuSH 29-mer shRNA constructs against PHF1 in pGFP-V-RS vector were purchased from OriGene, Inc.

Techniques: Transfection, Immunoprecipitation, Western Blot, Purification, Incubation, shRNA, Construct, Expressing, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: Polycomb Group Protein PHF1 Regulates p53-dependent Cell Growth Arrest and Apoptosis *

doi: 10.1074/jbc.M111.338996

Figure Lengend Snippet: PHF1 regulates cell proliferation and apoptosis in a p53-dependent manner. A, H1299, U2OS, HCT116+/+, and HCT116−/− were transfected with pcDNA3.1-PHF1 or control vector and selected with G418 for 2 weeks, and outgrowth colonies were stained by crystal violet. Representative photographs of cell colonies were shown. Total numbers of the colonies from three independent experiments were counted. An asterisk indicates statistical significance (*, p < 0.05; **, p < 0.001). B, two HuSH 29-mer shRNA constructs (KD-1 and KD-2) against PHF1 or control construct (NC) were transfected into cells, and colony formation assay was performed same as described in A. C, two HuSH 29-mer shRNA constructs (KD-1 and KD-2) against PHF1 or control construct (NC) were transfected into cells, at 72 h after transfection, the proliferative capacity of four cell lines was measured by BrdU ELISA assay. D, HCT116+/+ and HCT116−/− cells were transfected with PHF1 knockdown (KD1) and control shRNA (NC) constructs. At 72 h after transfection, the cells were treated with etoposide (40 μm). At 24 h after treatment, apoptosis was measured using PI staining assay. E, pcDNA3.0-p53 was co-transfected with shRNA constructs into HCT116−/− cells, and the proliferative capacity of HCT116−/− cells was measured by BrdU ELISA assay. F, pcDNA3.0-p53 was co-transfected with shRNA constructs into HCT116−/− cells, and etoposide-induced apoptosis was measured the same as D.

Article Snippet: RNA Interference The HuSH 29-mer shRNA constructs against PHF1 in pGFP-V-RS vector were purchased from OriGene, Inc.

Techniques: Transfection, Plasmid Preparation, Staining, shRNA, Construct, Colony Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Polycomb Group Protein PHF1 Regulates p53-dependent Cell Growth Arrest and Apoptosis *

doi: 10.1074/jbc.M111.338996

Figure Lengend Snippet: PHF1 expression was decreased in breast cancer specimens. Immunostaining of PHF1 in non-malignant mammary gland and primary ductal breast carcinoma. Table, quantification of PHF1 positive or PHF1-negative tissues.

Article Snippet: RNA Interference The HuSH 29-mer shRNA constructs against PHF1 in pGFP-V-RS vector were purchased from OriGene, Inc.

Techniques: Expressing, Immunostaining

Journal: The Journal of Biological Chemistry

Article Title: Polycomb Group Protein PHF1 Regulates p53-dependent Cell Growth Arrest and Apoptosis *

doi: 10.1074/jbc.M111.338996

Figure Lengend Snippet: PHF1 specifically activates p53 pathway. A, HepG2 cells were transfected with the indicated luciferase pathway reporter construct (100 ng), pTK-galactosidase (20 ng) construct and PHF1 expression construct (150 ng) or with the empty vector (150 ng). At 24 h after transfection, the luciferase activities were measured by luminometer. B, H1299, U2OS, HCT116 p53+/+, and HCT116 p53−/− cells were transfected, respectively, with the p53 responsive element reporter (p53-Luc, 100 ng) along with pTK-galactosidase (20 ng) and an increasing amount of PHF1 (100, 200 ng) and empty construct as indicated. At 24 h after transfection, the luciferase activities were measured by luminometer. C, HepG2 and U2OS cells were transfected with the p21 and Bax promoter reporters (p21-Luc/Bax-Luc, 100 ng), pTK-galactosidase (20 ng), and increasing amount of PHF1 (100 or 200 ng) or empty constructs as indicated. At 24 h after transfection, the luciferase activities were measured by luminometer. The mean value (± S.D.) of three independent experiments is shown.

Article Snippet: Antibodies The following antibodies were used in this work: PHF1 (sc-130646; Santa Cruz Biotechnology), PHF1 (AT3294a; Abgent), p53 (Do-1; sc-126; Santa Cruz Biotechnology), p53 (FL-393; sc-6243; Santa Cruz Biotechnology), acetyl-p53 (Lys-382; 2525S; Cell Signaling), phospho-Ser-15-p53 (9286S; Cell Signaling), p21 (Calbiochem), Myc (9E10; Sigma), FLAG (M2; Sigma), HA (MM5–101R; Convance), GFP (sc-8334; Santa Cruz Biotechnology), and actin (AC-74; Sigma).

Techniques: Transfection, Luciferase, Construct, Expressing, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Polycomb Group Protein PHF1 Regulates p53-dependent Cell Growth Arrest and Apoptosis *

doi: 10.1074/jbc.M111.338996

Figure Lengend Snippet: PHF1 interacts with p53 both in vivo and in vitro. A, H1299 cells were transfected with Myc-PHF1 and/or HA-p53 as indicated. Cell lysates were prepared and subjected to immunoprecipitation (IP) with anti-HA or Myc antibody. The immunoprecipitates were detected by Western blot (WB) with anti-Myc and anti-HA antibodies, respectively. B, co-immunoprecipitation of endogenous PHF1 and p53 proteins in U2OS cells. The endogenous p53 was immunoprecipitated by p53 (FL-393) antibody with rabbit IgG as a negative control. The immunoprecipitates were detected by Western blot with anti-PHF1 and p53 antibodies, respectively. C, H1299 cells were transfected with Myc-p53 and/or (&.) GFP-PHF1. Cells were fixed 48 h after transfection and subjected to immunofluorescence analysis. D, bacterially expressed GST fusion PHF1 protein bound to glutathione-Sepharose beads and incubated with His-tagged fusion p53 protein. Bound His-p53 was detected by Western blot with anti-His antibody. M.W., molecular weight.

Article Snippet: Antibodies The following antibodies were used in this work: PHF1 (sc-130646; Santa Cruz Biotechnology), PHF1 (AT3294a; Abgent), p53 (Do-1; sc-126; Santa Cruz Biotechnology), p53 (FL-393; sc-6243; Santa Cruz Biotechnology), acetyl-p53 (Lys-382; 2525S; Cell Signaling), phospho-Ser-15-p53 (9286S; Cell Signaling), p21 (Calbiochem), Myc (9E10; Sigma), FLAG (M2; Sigma), HA (MM5–101R; Convance), GFP (sc-8334; Santa Cruz Biotechnology), and actin (AC-74; Sigma).

Techniques: In Vivo, In Vitro, Transfection, Immunoprecipitation, Western Blot, Negative Control, Immunofluorescence, Incubation, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: Polycomb Group Protein PHF1 Regulates p53-dependent Cell Growth Arrest and Apoptosis *

doi: 10.1074/jbc.M111.338996

Figure Lengend Snippet: Delineation of the domains mediating the mutual interactions between PHF1 and p53. A, schematic presentation of p53 domains (left panel, top) and deletion mutants (left panel, bottom) and the results of GST pulldown experiments (right panel). Bacterially expressed GST fusion proteins of wild-type p53 and the deletion mutants were bound to glutathione-Sepharose beads, respectively, and incubated with lysates of H1299 cells transfected with a Myc-PHF1 expression construct. Bound Myc-PHF1 was detected by Western blot with anti-HA antibody, and various GST-p53 proteins were detected by ponceau S staining. B and C, schematic model of PHF1 domains (left panel, top) and deletion mutants (left panel, bottom) and the results of GST pulldown experiments (right panel). Bacterially expressed GST fusion proteins of wild-type PHF1 and the deletion mutants were bound to glutathione-Sepharose beads, respectively, and incubated with lysates of H1299 cells transfected with a Myc-p53 expression construct. Bound Myc-p53 was detected by Western blot with Do-1 antibody, and GST-PHF1 deletion mutant proteins were detected by ponceau S staining. M.W., molecular weight.

Article Snippet: Antibodies The following antibodies were used in this work: PHF1 (sc-130646; Santa Cruz Biotechnology), PHF1 (AT3294a; Abgent), p53 (Do-1; sc-126; Santa Cruz Biotechnology), p53 (FL-393; sc-6243; Santa Cruz Biotechnology), acetyl-p53 (Lys-382; 2525S; Cell Signaling), phospho-Ser-15-p53 (9286S; Cell Signaling), p21 (Calbiochem), Myc (9E10; Sigma), FLAG (M2; Sigma), HA (MM5–101R; Convance), GFP (sc-8334; Santa Cruz Biotechnology), and actin (AC-74; Sigma).

Techniques: Incubation, Transfection, Expressing, Construct, Western Blot, Staining, Mutagenesis, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: Polycomb Group Protein PHF1 Regulates p53-dependent Cell Growth Arrest and Apoptosis *

doi: 10.1074/jbc.M111.338996

Figure Lengend Snippet: PHF1 regulates p53 protein stability. A, the increasing amount of WT or various mutant expression constructs of Myc-PHF1 were co-transfected with same amounts of HA-p53 into H1299 cells. The protein levels of PHF1 and p53 were determined by Western blot. The pGFP-N3 expression construct was included as a transfection efficiency control. The quantification of immunoblot is shown in the lower panel. The mean value (± S.D.) of three independent experiments is shown. B, H1299 cells were co-transfected with HA-p53, along with empty vector, or Myc-PHF1 constructs. At 48 h after transfection, cells were treated with 30 μm cycloheximide (CHX) for the indicated lengths of time. Equal amounts of cell lysates were detected by Western blot, and for each experimental condition, the blot corresponds to one representative experiment, and the graph in the lower panel shows the quantification of p53 protein levels using GFP for standardization. The mean values (± S.D.) of three independent experiments are shown. C, U2OS cells and HepG2 cells were transiently transfected with two HuSH 29-mer shRNA constructs (KD-1 and KD-2) against PHF1 or control constructs (NC), and at 72 h after transfection, the protein levels of endogenous PHF1 and p53 were detected by Western blot. The quantification of immunoblots is shown in the right panel. The mean values (± S.D.) of three independent experiments are shown. D, qRT-PCR measurements of the mRNA levels of PHF1 and p53 in PHF1 knockdown U2OS cells. The left three columns are relative mRNA levels of PHF1, and the right three columns correspond to those of p53. The mRNA level of GAPDH was used for normalization. E, U2OS cells were transiently transfected with shRNA constructs against PHF1 (KD-1) or control constructs (NC), at 48 h after transfection, cells were treated with 30 μm cycloheximide (CHX) for the indicated lengths of time. Equal amounts of cell lysates were detected by Western blot. Right panel shows the quantification of p53 protein levels. F, qRT-PCR measurements of the expression of p53 target genes in control and PHF1 knockdown U2OS cells. The mean values (± S.D.) of three independent experiments are shown. G, U2OS cells transiently transfected with PHF1-specific shRNA (KD-1) or control shRNA (NC) were treated with 20 μm etoposide for the indicated time. Cell lysates were detected by Western blot with indicated antibodies.

Article Snippet: Antibodies The following antibodies were used in this work: PHF1 (sc-130646; Santa Cruz Biotechnology), PHF1 (AT3294a; Abgent), p53 (Do-1; sc-126; Santa Cruz Biotechnology), p53 (FL-393; sc-6243; Santa Cruz Biotechnology), acetyl-p53 (Lys-382; 2525S; Cell Signaling), phospho-Ser-15-p53 (9286S; Cell Signaling), p21 (Calbiochem), Myc (9E10; Sigma), FLAG (M2; Sigma), HA (MM5–101R; Convance), GFP (sc-8334; Santa Cruz Biotechnology), and actin (AC-74; Sigma).

Techniques: Mutagenesis, Expressing, Construct, Transfection, Western Blot, Plasmid Preparation, shRNA, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Polycomb Group Protein PHF1 Regulates p53-dependent Cell Growth Arrest and Apoptosis *

doi: 10.1074/jbc.M111.338996

Figure Lengend Snippet: PHF1 stabilizes p53 by protecting from MDM2-mediated ubiquitination and degradation. A, H1299 cells were transfected with Myc-PHF1 and/or FLAG-tagged ubiquitin ligases (MDM2, Pirh2, and COP1) and deubiquitinases (USP7 and USP10) as indicated. Cell lysates were prepared and subjected to immunoprecipitation with anti-FLAG antibody. The immunoprecipitates were eluted using 3× FLAG and detected by Western blot with indicated antibodies. B, bacterially expressed and purified GST-PHF1 or GST-PHF1-ΔBD1/2 were incubated with GST-p53 or GST-MDM2 in the presence of E1, E2 (UbcH5a), and ubiquitin. Following the ubiquitination reaction, the p53-ubiquitin conjugates were detected by Western blot with p53 antibody. C, HA-MDM2, pcDNA3.0-p53, Myc-PHF1, and FLAG-ubiquitin were co-transfected into H1299 cells. Cells were treated with of the proteasome inhibitor MG132 (20 μm) for 4 h before harvest. The polyubiquitinated forms of p53 were detected by Western blot with anti-p53 antibody. D, shRNA construct (KD-1) against PHF1 or control construct (NC) were co-transfected into U2OS cells. 48 h after transfection, the cells were treated with 20 μm etoposide for 8 h, the polyubiquitinated forms of endogenous p53 were detected by Western blot with anti-p53 antibody. Equal amount of p53 proteins in both control and PHF1 knockdown groups were loaded. E, HA-p53, FLAG-SUMO1, and Myc-PHF1 were co-transfected into H1299 cells. p53 was immunoprecipitated by anti-HA antibody. The sumoylated forms of p53 were detected by Western blot with anti-p53 antibody. F, the increasing amount of WT or ΔBD1/2 Myc-PHF1 expression constructs were co-transfected with HA-MDM2 and untagged p53 constructs into H1299 cells. The protein levels of PHF1, MDM2, and p53 were determined by Western blot. G, HA-MDM2, pcDNA3.0-p53, Myc-PHF1, or PHF1-ΔBD1/2 mutants were co-transfected into H1299 cells. 36 h after transfection, cells were lysed and subjected to immunoprecipitation using anti-p53 antibody. Immunoprecipitated proteins were detected with the indicated antibodies. H, HA-MDM2, FLAG-ubiquitin, and Myc-PHF1 constructs were co-transfected into H1299 cells. Cells were treated with MG132 for 4 h before harvest. MDM2 was immunoprecipitated by anti-HA antibody. The polyubiquitinated forms of MDM2 were detected by Western blot with anti-FLAG antibody. M.W., molecular weight.

Article Snippet: Antibodies The following antibodies were used in this work: PHF1 (sc-130646; Santa Cruz Biotechnology), PHF1 (AT3294a; Abgent), p53 (Do-1; sc-126; Santa Cruz Biotechnology), p53 (FL-393; sc-6243; Santa Cruz Biotechnology), acetyl-p53 (Lys-382; 2525S; Cell Signaling), phospho-Ser-15-p53 (9286S; Cell Signaling), p21 (Calbiochem), Myc (9E10; Sigma), FLAG (M2; Sigma), HA (MM5–101R; Convance), GFP (sc-8334; Santa Cruz Biotechnology), and actin (AC-74; Sigma).

Techniques: Transfection, Immunoprecipitation, Western Blot, Purification, Incubation, shRNA, Construct, Expressing, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: Polycomb Group Protein PHF1 Regulates p53-dependent Cell Growth Arrest and Apoptosis *

doi: 10.1074/jbc.M111.338996

Figure Lengend Snippet: PHF1 regulates cell proliferation and apoptosis in a p53-dependent manner. A, H1299, U2OS, HCT116+/+, and HCT116−/− were transfected with pcDNA3.1-PHF1 or control vector and selected with G418 for 2 weeks, and outgrowth colonies were stained by crystal violet. Representative photographs of cell colonies were shown. Total numbers of the colonies from three independent experiments were counted. An asterisk indicates statistical significance (*, p < 0.05; **, p < 0.001). B, two HuSH 29-mer shRNA constructs (KD-1 and KD-2) against PHF1 or control construct (NC) were transfected into cells, and colony formation assay was performed same as described in A. C, two HuSH 29-mer shRNA constructs (KD-1 and KD-2) against PHF1 or control construct (NC) were transfected into cells, at 72 h after transfection, the proliferative capacity of four cell lines was measured by BrdU ELISA assay. D, HCT116+/+ and HCT116−/− cells were transfected with PHF1 knockdown (KD1) and control shRNA (NC) constructs. At 72 h after transfection, the cells were treated with etoposide (40 μm). At 24 h after treatment, apoptosis was measured using PI staining assay. E, pcDNA3.0-p53 was co-transfected with shRNA constructs into HCT116−/− cells, and the proliferative capacity of HCT116−/− cells was measured by BrdU ELISA assay. F, pcDNA3.0-p53 was co-transfected with shRNA constructs into HCT116−/− cells, and etoposide-induced apoptosis was measured the same as D.

Article Snippet: Antibodies The following antibodies were used in this work: PHF1 (sc-130646; Santa Cruz Biotechnology), PHF1 (AT3294a; Abgent), p53 (Do-1; sc-126; Santa Cruz Biotechnology), p53 (FL-393; sc-6243; Santa Cruz Biotechnology), acetyl-p53 (Lys-382; 2525S; Cell Signaling), phospho-Ser-15-p53 (9286S; Cell Signaling), p21 (Calbiochem), Myc (9E10; Sigma), FLAG (M2; Sigma), HA (MM5–101R; Convance), GFP (sc-8334; Santa Cruz Biotechnology), and actin (AC-74; Sigma).

Techniques: Transfection, Plasmid Preparation, Staining, shRNA, Construct, Colony Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Polycomb Group Protein PHF1 Regulates p53-dependent Cell Growth Arrest and Apoptosis *

doi: 10.1074/jbc.M111.338996

Figure Lengend Snippet: PHF1 expression was decreased in breast cancer specimens. Immunostaining of PHF1 in non-malignant mammary gland and primary ductal breast carcinoma. Table, quantification of PHF1 positive or PHF1-negative tissues.

Article Snippet: Antibodies The following antibodies were used in this work: PHF1 (sc-130646; Santa Cruz Biotechnology), PHF1 (AT3294a; Abgent), p53 (Do-1; sc-126; Santa Cruz Biotechnology), p53 (FL-393; sc-6243; Santa Cruz Biotechnology), acetyl-p53 (Lys-382; 2525S; Cell Signaling), phospho-Ser-15-p53 (9286S; Cell Signaling), p21 (Calbiochem), Myc (9E10; Sigma), FLAG (M2; Sigma), HA (MM5–101R; Convance), GFP (sc-8334; Santa Cruz Biotechnology), and actin (AC-74; Sigma).

Techniques: Expressing, Immunostaining