Mouse monoclonal anti p53

The cells adherent on sterile glass coverslips were fixed in cold methanol for 20 min and permeabilized with 0.2% Triton X-100 in PBS1X for 10 min. Overnight incubation in cold room was performed with the following primary antibodies: rabbit polyclonal anti-GLUT1 (Millipore, Darmstadt, Germany; dilution 1:50); rabbit polyclonal anti-PTEN (Millipore; dilution 1: 200); mouse monoclonal anti-p53 (Santa Cruz Biotechnology, Santa Cruz, CA; dilution 1:100) and mouse monoclonal anti-LC3 (nanoTools, Teningen, Germany; dilution 1:100). As secondary antibody (dilution 1:600) the IRIS-2 (green fluorescence)-conjugated goat-anti-rabbit IgG secondary or IRIS-3 (red fluorescence)-conjugated goat-anti-mouse IgG (Cyanine Technologies SpA, Turin, Italy) was used as appropriate for 1 h at room temperature in a humid chamber. Nuclear chromatin was stained with the fluorescent dye 4,6-diamidino- 2-phenylindole-dihydrochloride (DAPI). As negative control, the primary antibody was omitted or substituted with pre-immune antiserum. Antibodies were diluted in PBS containing 0.1% Triton X-100 and 10% FBS. Stained cells were mounted with Slow-FADE (Light AntiFADE Kit, Molecular Probes Invitrogen, Carlsbad, CA, USA) and observed under a fluorescence microscope (Leica DMI6000B, Leica Microsystem AG, Wetzlad, Germany).

Cells were collected by centrifugation, washed with PBS, treated with RIPA lysis buffer, and sonicated. Whole-cell extracts were loaded onto a 10% SDS-polyacrylamide gel that was run at 150 V for 50 minutes in a Bio-Rad mini-gel system. Proteins were transferred to a nitrocellulose membrane (30 V, 90 minutes) and blocked for 1 h at room temperature in 5% BSA brought to 37°C or with nonfat milk at 4°C. Primary antibodies used were mouse monoclonal anti-p53 (Santa Cruz Biotechnology, Inc., TX, USA), rabbit monoclonal anti-phospho-p53 (Ser15) (Thermo Fisher Scientific, MA, USA), rabbit monoclonal anti-p21 (Abcam, UK), rabbit monoclonal anti-Bax (Abcam, UK), rabbit polyclonal anti-active caspase 3 (Abcam, UK), and mouse anti-GAPDH (Abcam, UK). Membranes were washed with TTBS (Tris 10 mM, NaCl 150 mM, and 0.005% Tween20) and incubated for 1 hour at room temperature with secondary antibody, goat anti-rabbit or goat anti-mouse, conjugated to horseradish peroxidase (Upstate/Millipore, MA, USA). Proteins were visualized using Immobilon Western kit (Upstate/Millipore, MA, USA) and the signal was captured with ChemiDoc XRS (Bio-Rad, CA, USA).

Cell lysis and Western blot analysis were performed as described previously [40 (link)], using the following antibodies: Rabbit polyclonal anti-SIK2 (Cell Signaling, Danvers, MA, USA), rabbit polyclonal anti-SIK2-pS385 (Kinexus, Sydney, Australia), rabbit polyclonal anti-AKT (Cell Signaling), rabbit polyclonal anti-AKT-pS473 (Cell Signaling), mouse monoclonal anti-β-actin (Sigma-Aldrich), rabbit polyclonal-anti KIF18B (ThermoFisher scientific, Waltham, MA, USA), rabbit polyclonal anti-EB1 (Abcam, Cambridge, UK), mouse monoclonal anti-cyclin B1 (Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal anti-Histone 3-pS10 (ThermoFisher scientific), mouse monoclonal anti-PLK1 (Santa Cruz Biotechnology), mouse monoclonal anti-vinculin (Santa Cruz Biotech), mouse monoclonal anti-P53 (Santa Cruz Biotech), rabbit polyclonal anti-P21 (cell signaling), rabbit polyclonal anti-BCL-xL (Santa Cruz Biotechnology), rabbit polyclonal anti-Cyclin D (Santa Cruz Biotechnology).
Reagents were purchased from the following sources: Paclitaxel (T7402) Sigma-Aldrich, propidium iodide (440300250) Acros Organics (Fair Lawn, NJ, USA), RNase A (1007885) Qiagen (Hilden, Germany), PE Annexin V (556421) and 7AAD (21-68981E) BD Biosciences, Caspase-Glo^® 3/7 Assay system (P1781) Promega, RO3306 (S7747) Selleckchem (Houston, TX, USA).

To evaluate the expression of proteins we used the following antibodies: mouse monoclonal anti-STAT3 (1:500) (BD Transduction Laboratories, 610,189), rabbit polyclonal anti-phospho-STAT3 (1:500) (p-Tyr705, clone D3A7, Cell Signaling Technology, 9145), mouse monoclonal anti-p53 (1:100) (clone DO-1, Santa Cruz Biotechnology Inc., sc-126), mouse monoclonal anti-HSP90 (1:100) (Santa Cruz Biotechnology Inc., sc-69703), mouse monoclonal anti-p21 (1:100) (clone F-8, Santa Cruz Biotechnology Inc., sc-271610), mouse monoclonal anti-SREBP1 (1:100) (clone A-4, Santa Cruz Biotechnology Inc., sc-365513), mouse monoclonal anti-MVK (1:100) (clone D-3, Santa Cruz Biotechnology Inc., sc-390669). Mouse monoclonal anti-β-actin (1:10,000) (Novus Biological, NB600-501) and goat polyclonal anti-lamin B (1:100) (Santa Cruz Biotechnology Inc, sc-374015) were used as loading control. The goat anti-mouse IgG-Horseradish Peroxidase (Santa Cruz Biotechnology Inc., sc- 2005), goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology Inc., sc-2004) and rabbit anti-goat IgG-HRP (Santa Cruz Biotechnology Inc., sc-2768) were used as secondary antibodies. All the primary and secondary antibodies were diluted in PBS-0.1% Tween20 solution containing 3% of BSA (SERVA).

At room temperature, 5 × 10⁶ cells were fixed with 1% formaldehyde (Sigma) for 10 min. Fixation was stopped with the addition of a 1/10 volume of 1.25 M glycine and with incubation for 5 min at room temperature. The sonication step was performed in a Qsonica sonicator (for 5 min alternating 20 s on and 20 s off) and 200 μg of the protein–chromatin complex was used for each round of IP. The antibody–protein complex was captured with preblocked Pierce Protein A/G Magnetic Beads. ChIP DNA was analyzed by qPCR with FastStart Universal SYBR Green Master (ROX) in an ABI 7500 using the following oligonucleotides: F: 5′-GCTGAGCCTTCGGGAACTGGCTTGC-3′ and R: 5′-CCGTTGGGCAGGAGCTGACGTCACC-3′. The following antibodies were used: 4.0 μg of mouse monoclonal anti-p53 (Santa Cruz, sc-126) and 4.0 μg of normal mouse IgG (Santa Cruz, sc-2025).

To evaluate protein expression on Western blot membranes, the following antibody were used: rabbit polyclonal anti-ATF6 (1:600) (Proteintech, 24169-1-AP), rabbit polyclonal anti-XBP1 (1:1000) (Novus Biologicals, NB100-80861), rabbit polyclonal anti-phospho-eIF2alpha (Ser51) (1:1000) (Cell Signaling, 9721), rabbit polyclonal anti-eIF2alpha (1:4000) (Cell Signaling, 9722), rabbit polyclonal anti-BiP/GRIP78 (1:5000) (Proteintech, 11587-1-AP), rabbit polyclonal anti-CHOP (GADD153) (1:1000) (Proteintech, 15204-1-AP), mouse monoclonal anti-DUSP5 (1:200) (Santa Cruz Biotechnology Inc., Dallas, TX, USA sc-393801), mouse monoclonal anti-p-ERK (Santa Cruz Biotechnology Inc., sc-7383), rabbit polyclonal anti-ERK1 (Santa Cruz Biotechnology Inc., sc-93), rabbit polyclonal anti-ERK2 (Santa Cruz Biotechnology Inc., sc-154), rabbit monoclonal anti-Mcl1 (1:1000) (Cell Signaling, 39224), mouse monoclonal anti-p53 (1:100) (clone DO-1, Santa Cruz Biotechnology Inc., sc-126). Mouse monoclonal anti-β-actin (1:10,000) (Sigma Aldrich) was used as the loading control. The goat antimouse IgG-HRP (1:30,000) (Bethyl Laboratories, A90-116P) and goat antirabbit IgG-HRP (1:30,000) (Bethyl Laboratories, A120-101P) were used as secondary antibodies. All primary and secondary antibodies were diluted in PBS–0.1% Tween20 solution containing 3% of BSA (SERVA).

To evaluate the expression of proteins, we used the following antibodies: mouse monoclonal anti-PARP1 (1:100) (Santa Cruz Biotechnology Inc.), mouse monoclonal anti-p53 (1:100) (clone DO-1, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-p21 (1:100) (Santa Cruz Biotechnology Inc.), mouse monoclonal anti-MVK (1:100) (Santa Cruz Biotechnology Inc.), mouse monoclonal anti-pERK (1:200) (Santa Cruz Biotechnology Inc.), rabbit polyclonal anti-ERK1 and anti-ERK2 (1:200) (Santa Cruz Biotechnology Inc.), rabbit polyclonal anti-BIP (1:1000) (Cell Signaling) and mouse monoclonal anti-CHOP (1:100) (Santa Cruz Biotechnology Inc.). Mouse monoclonal anti-β-actin (1:10,000) (Novus Biological) was used as loading control. The goat anti-mouse IgG-Horseradish Peroxidase (Santa Cruz Biotechnology Inc.) and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology Inc.) were used as secondary antibodies. All the primary and secondary antibodies were diluted in PBS—0.1% Tween20 solution containing 3% of BSA (SERVA).