primary polyclonal antibodies for p53  (Bioss)

Journal: Microbiology Spectrum

Article Title: A Critical Role for p53 during the HPV16 Life Cycle

doi: 10.1128/spectrum.00681-22

Figure Lengend Snippet: Tumor suppressor p53 expression is conserved in HPV16 immortalized cell lines and patient derived xenografts. (A) Western blot analysis of p53 and pRb in N/tert-1 cells stably expressing the HPV16 genome (lane 2), HPV16 E6+E7 (lane 4) or empty vector (lane 1) compared to Human tonsillar keratinocytes immortalized with HPV16 (lane 3). Two independent human foreskin keratinocyte (HFK) donors were immortalized with wild-type HPV16 (lanes 6 and 7) or exogenous expression of the HPV16 E6 and E7 oncogenes (lane 8, cells from donor 1). (B) Western blot analysis of p53 expression in 4 HPV-negative and 4 HPV-positive patient derived xenografts. (C) p53 expression following 24-h cisplatin treatment of primary HPV-positive head and neck cancer cell lines (SCC47 and SCC104) DMF solvent only was used as drug-free control. All Western blots utilized GAPDH as an internal loading control.

Article Snippet: Primary polyclonal antibody against p53 (Invitrogen; PA5-27822) or a HA-tag antibody (used as a negative control) was incubated in 200 μg of cell lysate (prepared as described above), made up to a total volume of 500 μL with lysis buffer (0.5% Nonidet P-40, 50 mM Tris [pH 7.8], and 150 mM NaCl), supplemented with protease inhibitor (Roche Molecular Biochemicals) and phosphatase inhibitor cocktail (Sigma) and rotated at 4°C overnight.

Techniques: Expressing, Derivative Assay, Western Blot, Stable Transfection, Plasmid Preparation

Journal: Microbiology Spectrum

Article Title: A Critical Role for p53 during the HPV16 Life Cycle

doi: 10.1128/spectrum.00681-22

Figure Lengend Snippet: p53 reduction in via introduction of full-length HPV16 E6 reduces cellular proliferation in HPV16 immortalized foreskin keratinocytes. (A) 11-day growth curve of N/Tert-1 cells expressing exogenous HPV16 E6 compared to empty vector. (B) 13-day growth curve of human foreskin keratinocytes immortalized by HPV16 and stably expressing exogenous full-length E6, mutant E6 that does not bind and degrade p53 (E6Δp53) or GFP control vector. (C) Senescence staining of cells in B at day 11. Arrows indicate positively staining cells. (D) Quantification of senescence staining in C as percent positively stained per field. Western blot analysis of p53 expression following transfection of E6 plasmids (day 0) and after growth rate recovery of HFK+HPV16+E6 (day 13). (E) Western blot analysis of p53 in HFKs with exogenous E6 and E6Δp53 expression at day 0 and day 13. GAPDH was used as internal loading control. (F) RT-qPCR analysis of exogenous GFP, E6 and E6Δp53 expression at day 0 and day 13 using primers against FLAG-HA tag. Relative quantity calculated by the ΔΔC T method using GAPDH as an internal control. Bonferroni correction utilized when applicable. (G) Western blotting of the indicated extracts using FLAG antibody (the E6 is double tagged with HA and FLAG).

Article Snippet: Primary polyclonal antibody against p53 (Invitrogen; PA5-27822) or a HA-tag antibody (used as a negative control) was incubated in 200 μg of cell lysate (prepared as described above), made up to a total volume of 500 μL with lysis buffer (0.5% Nonidet P-40, 50 mM Tris [pH 7.8], and 150 mM NaCl), supplemented with protease inhibitor (Roche Molecular Biochemicals) and phosphatase inhibitor cocktail (Sigma) and rotated at 4°C overnight.

Techniques: Expressing, Plasmid Preparation, Stable Transfection, Mutagenesis, Staining, Western Blot, Transfection, Quantitative RT-PCR

Journal: Microbiology Spectrum

Article Title: A Critical Role for p53 during the HPV16 Life Cycle

doi: 10.1128/spectrum.00681-22

Figure Lengend Snippet: Generation and characterization of p53 binding mutant of HPV16 E2 (E2-p53) in N/Tert-1 cells. (A) Input Western blot of stably expressing E2 and E2(-p53) in N/Tert-1 Cells. For E2(-p53), residues W341, D344 and D338 were mutated to alanine as previously described ( , ). (B) Co-immunoprecipitation pull down of E2 using polyclonal antibody against p53. (C) HPV16 long control region repression assay of wild-type E2 and E2(-p53). N/Tert-1 cells were transiently transfected with 1 μg pHPV16-LCR-Luciferase reporter plasmid along with 10 ng, 100 ng, or 1000 ng of E2 or E2(-p53) plasmid. (D) E2 transcriptional activity assay of wild-type E2 and E2(-p53). Similar to LCR repression assay, N/Tert-1 cells were transiently transfected with 1 μg pTK6E2-Luciferase reporter plasmid along with increasing amounts of E2 wild-type and E2(-p53) plasmids. For (C) and (D), relative luminescence units were calculated by normalizing absolute luminescence readouts to input protein concentration. (E) U2OS cells stably expressing E2-WT and E2(-p53) were generated and a p53 co-immunoprecipitation carried out. Left panel, input; right panel, co-IP. (F) Transient DNA replication assays were carried out on U2OS cells transfected with pOri, E1 or the indicated E2. Both E1+E2-WT and E1+E2(-p53) increased replication similarly, both significantly above background. (G) HA tagged E1 was transfected into the indicated cell lines and a HA co-immunoprecipitation carried out. Left panel, input; right panel, co-IP. (H) Southern blot of SphI digested DNA (cuts the HPV16 genome once) from the indicated immortalized human foreskin keratinocytes. An over exposure of this blot indicated a band in Donor 2 wild-type cells that migrated around 7.5kbp, indicating a part of the genome may have been lost during immortalization. PCR demonstrates that viral DNA is in these cells, and they are immortalized. With donor 1 there is less DNA with the mutant genome than the wild type, the opposite of Donor 2. Therefore, the mutation did not trend toward influencing the levels of DNA in the immortalized HFK. (I) TV exonuclease digestion assay to determine viral genome status. We looked at GAPDH in this assay and called the ΔCt for GAPDH 100% degradation, then we estimated the resistance of both mitochondrial (mito) DNA and HPV16 (E6) to degradation. In all cases the HPV16 DNA is predominantly episomal. As an example, if the ΔCt for GAPDH was 10 following exonuclease treatment, and the ΔCt for mito and E6 equals 1, then they were estimated as 90% episomal DNA (mitochondria have circular genomes that are resistant to the exonuclease). Low pass indicates low passage, 7 or less. High pass indicates high passage, 12 or greater. This demonstrates that, even following prolonged culture, there is no shift toward integration of the HPV16 genomes. The results shown are from duplicate or triplicate experiments, and standard error bars are shown.

Article Snippet: Primary polyclonal antibody against p53 (Invitrogen; PA5-27822) or a HA-tag antibody (used as a negative control) was incubated in 200 μg of cell lysate (prepared as described above), made up to a total volume of 500 μL with lysis buffer (0.5% Nonidet P-40, 50 mM Tris [pH 7.8], and 150 mM NaCl), supplemented with protease inhibitor (Roche Molecular Biochemicals) and phosphatase inhibitor cocktail (Sigma) and rotated at 4°C overnight.

Techniques: Binding Assay, Mutagenesis, Western Blot, Stable Transfection, Expressing, Immunoprecipitation, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Protein Concentration, Generated, Co-Immunoprecipitation Assay, Southern Blot

Journal: Microbiology Spectrum

Article Title: A Critical Role for p53 during the HPV16 Life Cycle

doi: 10.1128/spectrum.00681-22

Figure Lengend Snippet: Generation and characterization of HPV16-p53 immortalized human foreskin keratinocytes (HFKs). p53 protein expression in two independent HFK donors immortalized by wild-type HPV16 (Lanes 3 and 5) and HPV16(-p53) (Lanes 2 and 4). N/Tert-1 and HFK immortalized by E6 and E7 are provided for reference (lanes 1 and 6, respectively). All lines were grown as pools. Activation of the ATR and ATM DNA-damage pathways in immortalized HFKs. ATR and ATM activation by HPV16 leads to phosphorylation of Checkpoint kinases 1 and 2, respectively, and serve as markers for HPV infection and replication.

Article Snippet: Primary polyclonal antibody against p53 (Invitrogen; PA5-27822) or a HA-tag antibody (used as a negative control) was incubated in 200 μg of cell lysate (prepared as described above), made up to a total volume of 500 μL with lysis buffer (0.5% Nonidet P-40, 50 mM Tris [pH 7.8], and 150 mM NaCl), supplemented with protease inhibitor (Roche Molecular Biochemicals) and phosphatase inhibitor cocktail (Sigma) and rotated at 4°C overnight.

Techniques: Expressing, Activation Assay, Infection