mouse monoclonal anti mdm2 smp14  (Santa Cruz Biotechnology)

Journal: Cell reports

Article Title: Rucaparib Treatment Alters p53 Oscillations in Single Cells to Enhance DNA-Double-Strand-Break-Induced Cell Cycle Arrest

doi: 10.1016/j.celrep.2020.108240

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following antibodies were used: mouse monoclonal anti-p53 DO-1 (Santa Cruz Biotechnology, sc-126) [1:1,000], mouse monoclonal anti-MDM2 SMP14 (Santa Cruz, sc-965) [1:500], rabbit polyclonal anti-WIP1 H-300 (Santa-Cruz,sc-20712), rabbit polyclonal anti- phospho-DNA-PKcs S2056 (Abcam,ab18192) [1:1000], mouse monoclonal anti-DNA-PKcs (Abcam, ab44815)[1:200], rabbit monoclonal anti-phospho-ATM S1981 (Cell Signaling Technology, 5883)[1:1000], rabbit monoclonal anti-phospho-Chk2 T68 (Cell Signaling Technology, 2197)[1:1000], rabbit monoclonal anti-β-Actin (Cell Signaling Technology, 4970)[1:3000], mouse monoclonal against γH2AX (Millipore, 05-636)[1:200].

Techniques: Recombinant, Blocking Assay, Expressing, Software

Journal: Communications Biology

Article Title: Tau affects P53 function and cell fate during the DNA damage response

doi: 10.1038/s42003-020-0975-4

Figure Lengend Snippet: a , b Parental (wt) or 232P (Tau-KO) cells treated 30 min without (basal) or with 60 μM etoposide and recovered for 6 h in the absence (eto) or presence of 10 µg/mL KU-55933 and/or 5 µg/mL nutlin-3. a Mean intensity ± sem of single-cell nuclear P53 or MDM2 (DAPI mask, ImageJ) shown as fold of basal conditions, n > 100 cells/condition distributed over five images. b Percent clCasp3-positive cells shown as mean ± SD of five images (basal) or 15 images (treatments), n > 500 cells/condition. Non-parametric independent samples test and Kruskal–Wallis pairwise comparison between cell lines (in bold) or for treatment for each cell line (in vertical). c Western bot analysis of P53 in parental (wt) or 232 P (Tau-KO) cells at basal conditions, after 30 min 60 μM etoposide and 4 h recovery without or with 10 μM MG132. GAPDH served as loading control. d Parental (wt) or 232P (Tau-KO) cells pre-treated for 30 min with 60 μM etoposide followed by 4 h with 10 μM MG132, were incubated with 25 μM of cycloheximide (CHX) for the indicated chase times. Single-cell nuclear P53 or nuclear MDM2 (DAPI mask, ImageJ) shown as fold of wt cells at basal conditions. Mean intensity ± sem of n > 100 cells/condition distributed over five images. Independent measures ordinary two-way ANOVA, source of variation for cell lines (bold), multiple Bonferroni pairwise comparisons of treatment for each line (in italic). e Parental (wt) or (Tau-KO) 232P cells treated for 30 min with 60 μM etoposide and 6 h recovery analyzed with a 90 kDa MDM2 rabbit antibody (green and middle panel with GAPDH as loading control) or a 60 and 90 kDa MDM2 mouse antibody (red and bottom panel).

Article Snippet: The cell lysates for P53 immune precipitation were cleared by centrifugation at 20,000 g per 10 min. For immune blots , we used 0.2 μg/mL Tau13 (sc-21796, Santa Cruz), 0.18 μg/mL GAPDH (ab181602, Abcam), 0.4 μg/mL P53 DO-1 (sc-126, Santa Cruz), 4 μg/mL P53 Pab 1801 (sc-98, Santa Cruz), 0.5 μg/mL pS 15 -P53 (ab223868, Abcam), 0.1 μg/mL rabbit D1V2Z MDM2 (#86934, Cell Signaling), 0.2 μg/mL mouse SMP14 MDM2 (sc965, Santa Cruz), 0.05 μg/mL clAsp 175 -Caspase3 (#9661, Cell Signaling), or 0.2 μg/mL p21 (sc53870, Santa Cruz).

Techniques: Comparison, Western Blot, Control, Incubation

Journal: Communications Biology

Article Title: Tau affects P53 function and cell fate during the DNA damage response

doi: 10.1038/s42003-020-0975-4

Figure Lengend Snippet: Cell lysates of SH-SY5Y treated with 10 μM of MG132 to stabilize P53 expression, without (ctrl) or with (eto) a 30 min pre-treatment with 60 μM etoposide, were subjected to immune precipitation of endogenous P53 with a rabbit antibody (P53) or with a rabbit GFP antibody as negative IP control (GFP). Western blot analysis for co-precipitation of MDM2 or Tau with the respective mouse antibodies as indicated. The blots on the top show the analysis of the starting material (cell lysates), those on the bottom the immunoprecipitation (IP). The P53 blots are entirely shown, whereas for MDM2 and Tau, the blots were cut between the 55 kDa and the 95 kDa protein size markers and analyzed separately.

Article Snippet: The cell lysates for P53 immune precipitation were cleared by centrifugation at 20,000 g per 10 min. For immune blots , we used 0.2 μg/mL Tau13 (sc-21796, Santa Cruz), 0.18 μg/mL GAPDH (ab181602, Abcam), 0.4 μg/mL P53 DO-1 (sc-126, Santa Cruz), 4 μg/mL P53 Pab 1801 (sc-98, Santa Cruz), 0.5 μg/mL pS 15 -P53 (ab223868, Abcam), 0.1 μg/mL rabbit D1V2Z MDM2 (#86934, Cell Signaling), 0.2 μg/mL mouse SMP14 MDM2 (sc965, Santa Cruz), 0.05 μg/mL clAsp 175 -Caspase3 (#9661, Cell Signaling), or 0.2 μg/mL p21 (sc53870, Santa Cruz).

Techniques: Expressing, Control, Western Blot, Immunoprecipitation

Journal: Communications Biology

Article Title: Tau affects P53 function and cell fate during the DNA damage response

doi: 10.1038/s42003-020-0975-4

Figure Lengend Snippet: PCR Primers (all specific for homo sapiens mRNAs).

Article Snippet: The cell lysates for P53 immune precipitation were cleared by centrifugation at 20,000 g per 10 min. For immune blots , we used 0.2 μg/mL Tau13 (sc-21796, Santa Cruz), 0.18 μg/mL GAPDH (ab181602, Abcam), 0.4 μg/mL P53 DO-1 (sc-126, Santa Cruz), 4 μg/mL P53 Pab 1801 (sc-98, Santa Cruz), 0.5 μg/mL pS 15 -P53 (ab223868, Abcam), 0.1 μg/mL rabbit D1V2Z MDM2 (#86934, Cell Signaling), 0.2 μg/mL mouse SMP14 MDM2 (sc965, Santa Cruz), 0.05 μg/mL clAsp 175 -Caspase3 (#9661, Cell Signaling), or 0.2 μg/mL p21 (sc53870, Santa Cruz).

Techniques:

Journal: bioRxiv

Article Title: Acetylation-mediated phase control of the nucleolus regulates cellular acetyl-CoA responses

doi: 10.1101/2020.01.24.918706

Figure Lengend Snippet: (A) Immunoblotting for p53 in ASA-WT and ASA-KO cells cultured in 10% or 1% FBS containing media with or without acetate for 4 hours. Histone H3 is shown as a loading control. (B) Schematic representation of the nucleolus as an organelle for ribosomal biogenesis (left) and the stressed nucleolus that induces p53 stabilization through the inhibitory interaction between the 5S rRNA-RPL11-RPL5 and MDM2. (C) Immunoprecipitation with FLAG-RPL11 and Immunoblotting for MDM2 and FLAG-RPL11 in ASA-KO cells cultured in 1% FBS containing media with or without acetate for the indicated hours. (D) Immunoblotting for p53 levels in ASA-KO cells pretreated with indicated siRNAs and cultured in 1% FBS containing media with or without acetate for 4 hours. Histone H3 is shown as a loading control.

Article Snippet: Antibodies were sourced as indicated: ATP-Citrate Lyase Rabbit polyAb (1:1000, Cell Signaling Technology, #4332) Histone H3 (D1H2) XP ® Rabbit mAb (1:1000, Cell Signaling Technology, #4499) Acetyl-Histone H3 (Lys9) (C5B11) Rabbit mAb (1:1000, Cell Signaling Technology, #9649) Acetyl-Histone H3 (Lys27) (D5E4) XP ® Rabbit mAb (1:1000, Cell Signaling Technology, #8173) Acetyl-Histone H4 (Lys8) Rabbit polyAb (1:1000, Cell Signaling Technology, #2594) Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (1:1000 or 1:200, Cell Signaling Technology, #9718) Histone H2A (D6O3A) Rabbit mAb (1:1000, Cell Signaling Technology, #12349) Acetylated-Lysine Antibody Rabbit polyAb (1:1000, Cell Signaling Technology, #9814) α-Tubulin (clone DM1A) Mouse mAb (1:5000, Sigma, T6199) Acetyl-α-Tubulin (Lys40) (D20G3) XP ® Rabbit mAb (1:1000, Cell Signaling Technology, #5335) p53 (DO-1) Mouse mAb (1:5000, Santa Cruz, sc-126) GLTSCR2/PICT1 Rabbit polyAb (1:1000, Cell Signaling Technology,73225) MDM2 (SMP14) Mouse mAb (1:1000, Santa Cruz, sc-965) RPL11 (D1P5N) Rabbit polyAb (1:1000, Cell Signaling Technology,18163) RPL5 Rabbit polyAb (1:1000, Cell Signaling Technology,14568) anti-BrdU [BU1/75 (ICR1)] Rat mAb (1:500, abcam, ab6326) BOP1 (E-1) Mouse mAb (1:250, Santa Cruz, sc-390672) RRP1 Rabbit polyAb (1:200, GeneTex, Irvine, CA, GTX115107) UBF (F-9) Mouse mAb (1:500, Santa Cruz, sc-13125) FBL (G-8) Mouse mAb (1:500, Santa Cruz, sc-374022) FBL (B-1) Mouse mAb (1:500, Santa Cruz, sc-166001) NCL/C23 (H-6) Mouse mAb (1:500, Santa Cruz, sc-55486) NCL (D4C7O) Rabbit mAb (1:1000, Cell Signaling Technology, #14574) NPM1 Rabbit polyAb (1:200,, Cell Signaling Technology, #3542) Anti-FLAG M2 Mouse mAb (1:1000, Sigma, F3165)

Techniques: Western Blot, Cell Culture, Control, Immunoprecipitation

Journal: Cancer research

Article Title: Peli1 modulates the subcellular localization and activity of Mdmx

doi: 10.1158/0008-5472.CAN-17-3531

Figure Lengend Snippet: (A) Silver staining of affinity-purified Mdmx complexes from the cell extracts of the FH-Mdmx A375 stable cells and the parental A375 cells. Mdmx-interacting partners were identified by mass spectrometry, and the Peli1 peptide sequences are presented.

Article Snippet: Mouse monoclonal antibody for Peli1 (F-7), p53 (DO-1), Mdm2 (SMP14) and Myc (9E10) and rabbit polyclonal antibodies for p21 (C19) and Puma (H-136) were purchased from Santa Cruz Biotechnology.

Techniques: Silver Staining, Affinity Purification, Mass Spectrometry

Journal: Cancer research

Article Title: Peli1 modulates the subcellular localization and activity of Mdmx

doi: 10.1158/0008-5472.CAN-17-3531

Figure Lengend Snippet: (A) A schematic representation of Mdmx and the corresponding mutants (+: binding positive to Peli1; -: binding negative to Peli1).

Article Snippet: Mouse monoclonal antibody for Peli1 (F-7), p53 (DO-1), Mdm2 (SMP14) and Myc (9E10) and rabbit polyclonal antibodies for p21 (C19) and Puma (H-136) were purchased from Santa Cruz Biotechnology.

Techniques: Binding Assay

Journal: Cancer research

Article Title: Peli1 modulates the subcellular localization and activity of Mdmx

doi: 10.1158/0008-5472.CAN-17-3531

Figure Lengend Snippet: (A) Peli1 cannot bind to p53. Whole cell extracts or immunoprecipitates with FLAG/M2 beads from H1299 cells transfected with Myc-Peli1 alone or together with FH-Mdmx or FLAG-p53 were subjected to western blot with anti-Myc (top) and anti-FLAG antibody (lower).

Article Snippet: Mouse monoclonal antibody for Peli1 (F-7), p53 (DO-1), Mdm2 (SMP14) and Myc (9E10) and rabbit polyclonal antibodies for p21 (C19) and Puma (H-136) were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Western Blot

Journal: Cancer research

Article Title: Peli1 modulates the subcellular localization and activity of Mdmx

doi: 10.1158/0008-5472.CAN-17-3531

Figure Lengend Snippet: (A–B) U2OS Peli1 inducible stable line cells were transfected with control siRNA or siRNA against p53 or Mdmx. After 24 hours, the cells were without treatment or treated with 0.1 μg/ml of doxycycline 48 hours. (A) Western blot analysis of cell extracts with antibody against Peli1, Mdm2, Mdmx, p53, p21 and β-actin. (B) p21 and Mdm2 mRNA levels were determined by real-time PCR in these cells. (C) Western blot analysis of cell extracts from two U2OS Peli1 CRISPR-Cas9 knock-out cell lines (CRISPR 1 and 2) with antibody against Mdm2, Mdmx, p53, Peli1, p21 and β-actin. Parental U2OS cells were used as the control. (D) p21 and Mdm2 mRNA levels were determined by real-time PCR in U2OS Peli1 knock-out cell lines. Parental U2OS cells were used as the control.

Article Snippet: Mouse monoclonal antibody for Peli1 (F-7), p53 (DO-1), Mdm2 (SMP14) and Myc (9E10) and rabbit polyclonal antibodies for p21 (C19) and Puma (H-136) were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Control, Western Blot, Real-time Polymerase Chain Reaction, CRISPR, Knock-Out

Journal: Cancer research

Article Title: Peli1 modulates the subcellular localization and activity of Mdmx

doi: 10.1158/0008-5472.CAN-17-3531

Figure Lengend Snippet: (A) FH-Mdmx was transfected alone, or with Myc-Peli1, or with Myc-Peli1 mutant (C395/398A) in H1299 cells. Myc-Peli1 or Myc-Peli1 mutant were also individually transfected into the cells. Twenty-four hours after transfection, the cells were fixed, permeabilized and then incubated with anti-Myc mouse antibody or anti-HA rat antibody followed by Alexa Fluor 488 (green) conjugated anti-mouse and Alexa Fluor 568 (red) conjugated anti-rat secondary antibody. DAPI was used for the nuclear staining. The representative images of Mdmx, Peli1, DAPI and merged staining are shown.

Article Snippet: Mouse monoclonal antibody for Peli1 (F-7), p53 (DO-1), Mdm2 (SMP14) and Myc (9E10) and rabbit polyclonal antibodies for p21 (C19) and Puma (H-136) were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Mutagenesis, Incubation, Staining

Journal: Cancer research

Article Title: Peli1 modulates the subcellular localization and activity of Mdmx

doi: 10.1158/0008-5472.CAN-17-3531

Figure Lengend Snippet: (A) Kaplan-Meier survival curves of Peli1 wild-type (n=11), Peli1 null (n=18), Peli1 wild-type and Eμ-Myc (n=31) and Peli1 null and Eμ-Myc mice (n=29). Statistical significance was determined using log-rank test (p<0.01; Peli1 wild-type versus null mice in Eμ-Myc background).

Article Snippet: Mouse monoclonal antibody for Peli1 (F-7), p53 (DO-1), Mdm2 (SMP14) and Myc (9E10) and rabbit polyclonal antibodies for p21 (C19) and Puma (H-136) were purchased from Santa Cruz Biotechnology.

Techniques: