Journal: Infection and Immunity

Article Title: The Fimbrial Protein FlfA from Gallibacterium anatis Is a Virulence Factor and Vaccine Candidate

doi: 10.1128/IAI.00059-13

Figure Lengend Snippet: Bacterial strains and plasmids used in this study

Article Snippet: The bacterial strains and plasmids used in the present study are listed in . table ft1 table-wrap mode="anchored" t5 caption a7 Strain or plasmid Relevant characteristics a Source or reference Strains G. anatis 12656-12 Wild-type strain; biovar 4, isolated in 1981 (Denmark) 51 Δ flfA mutant flfA deletion mutant of G. anatis 12656-12; Km r This study F149 T Wild-type strain; isolated in 1979 (Denmark) 2 10672/6 Wild-type strain; biovar 1 (Denmark) 2 4895 Wild-type strain; biovar 4 (Mexico) 52 7990 Wild-type strain; biovar 1 (Mexico) 52 Avicor Wild-type strain; biovar 3 (Mexico) 52 CCM5995 Wild-type strain; biovar 20, isolated in 1978 (Czech Republic) 2 IPDH 697-78 Wild-type strain; biovar 15, isolated in 1978 (Germany) 2 E. coli TOP10 E. coli host strain for routine cloning Invitrogen DH5α E. coli host strain for routine cloning Invitrogen BL21 CodonPlus E. coli strain for expression of recombinant proteins Stratagene Plasmids pENTR/SD/D-TOPO Directional cloning vector for entry to the Gateway System Invitrogen pDEST17 Gateway Destination vector (Amp r , N-terminal His 6 fusion tag) Invitrogen pDEST17 flfA FlfA expression vector (Amp r , N-terminal His 6 fusion tag) This study pBluescript II KS(+) General purpose cloning vector (Amp r ) Stratagene pUC4K Vector carrying the Km r cassette from Tn 903 53 pBSΔ flfA pBluescript II KS(+) containing flanking regions of the flfA gene (Amp r ) This study pBSΔ flfA -Km r Plasmid for flfA deletion with flanking regions of the gene cloned on either side of the Km r cassette from pUC4K into pBluescript II KS(+) (Amp r Km r ) This study Open in a separate window a Amp r , ampicillin resistance; Km r , kanamycin resistance; His, histidine.

Techniques: Plasmid Preparation, Isolation, Mutagenesis, Clone Assay, Expressing, Recombinant

Journal: Oncogene

Article Title: p53 inhibits mRNA 3' processing through its interaction with the CstF/BARD1 complex.

doi: 10.1038/onc.2011.29

Figure Lengend Snippet: Figure 1 CstF1 interacts with both BARD1 and p53 to form a protein complex. (a) Diagram of BARD1, CstF1 and p53 derivatives. (b) Requirement of p53 C-terminal domain for both CstF1 and BARD1 interaction. Recombinant His-p53 or the indicated His-p53 derivatives were incubated with purified GST, GST-CstF1 or GST-BARD1. Protein samples were treated with RNase A. Bound proteins were eluted and analyzed by western blot with anti-p53 antibodies. Five percent of His-p53 or His-p53 derivatives used in the reaction are shown as input. (c) CstF1, BARD1 and p53 can coexist in complexes. Either GST-BARD1 or GST-CstF1 was immobilized in glutathione-Sepharose beads to test its ability to bind CstF1/BARD1 and p53 from NE of HeLa cells, while increasing concentrations of recombinant His-p53 (5, 10 and 20 ng) were added. Bound proteins were detected by immunoblotting with CstF1 and p53 antibodies. The pellets (PD) and the supernatant (SN) were analyzed. (d) p53, CstF and BARD1 co-immunoprecipitate from NEs of MCF7 cells treated with UV irradiation. CstF and p53 co-immunoprecipitation is irrespective of UV irradiation. Co-immunoprecipitation assays were performed using NEs of cells exposed or not to UV irradiation and allowed to recover for 2 h as described before. NEs were immunoprecipitated with either anti-CstF2, to ensure that any detected interactions were between p53 and intact CstF, or anti-p53 antibodies. Protein samples were treated with RNase A. Equivalent amounts of the pellets (IP) and the supernatants (SN) were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and proteins were detected by immunoblotting with antibodies against BARD1, CstF2 and p53. Antibodies against Topo II were used as a control of specificity. Positions of Topo II, CstF2 and p53 are indicated. Twenty percent of the NE used in the immunoprecipitation reaction is shown as input.

Article Snippet: Sixty micrograms of each NE was analyzed by immunoblotting with antibodies against BARD1 (H-300, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53 (SC-126, Santa Cruz), CstF2 (generously provided by Dr Manley, Columbia University, New York, NY, USA), CstF1 (10064-2-AP, Protein Tech Group, Chicago, IL, USA) and Topoisomerase II (Topo II, SC-25330, Santa Cruz). siRNA knockdown of p53 expression in HeLa and MCF7 cells The siRNA specific for human p53 was synthesized by Qiagen (Valencia, CA, USA) and the control RNA duplex used as non-silencing siRNA was obtained from Dharmacon RNA Technologies (Lafayette, CO, USA).

Techniques: Recombinant, Incubation, Western Blot, Irradiation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Control

Journal: Oncogene

Article Title: p53 inhibits mRNA 3' processing through its interaction with the CstF/BARD1 complex.

doi: 10.1038/onc.2011.29

Figure Lengend Snippet: Figure 2 The C-terminal domain of p53 inhibits 30 cleavage in vitro and the levels of 30 cleavage correlate with expression levels of p53. (a) The C-terminal domain of p53 is necessary for inhibition of 30 cleavage. NEs from HeLa cells were preincubated with no addition or increasing amounts of the indicated His-p53 derivatives or GST proteins (40, 80 and 120 ng). After 15 min, L3 pre-mRNA was added and incubation continued for 90 min. RNAs were purified and analyzed by denaturing PAGE. Positions of pre-mRNA and the 50 cleavage product are indicated. (b) siRNA-mediated knockdown of p53 affects the UV-induced inhibition of 30 cleavage in HeLa cells. NEs from cells treated with p53/control siRNA and UV irradiation, and allowed to recover for 2 h, were analyzed for L3 pre-mRNA 30 cleavage. Positions of pre-mRNA and the 50 cleavage product are indicated. (c) siRNA-mediated knockdown of p53 induces 30 cleavage in MCF7 cells. NEs from cells were analyzed as described in (b). (d) Comparison of p53 expression levels in NE from HeLa and MCF7 cells. Protein concentrations in NE from siRNA treated/untreated cells were normalized by immunostaining with antibodies against Topo II.

Article Snippet: Sixty micrograms of each NE was analyzed by immunoblotting with antibodies against BARD1 (H-300, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53 (SC-126, Santa Cruz), CstF2 (generously provided by Dr Manley, Columbia University, New York, NY, USA), CstF1 (10064-2-AP, Protein Tech Group, Chicago, IL, USA) and Topoisomerase II (Topo II, SC-25330, Santa Cruz). siRNA knockdown of p53 expression in HeLa and MCF7 cells The siRNA specific for human p53 was synthesized by Qiagen (Valencia, CA, USA) and the control RNA duplex used as non-silencing siRNA was obtained from Dharmacon RNA Technologies (Lafayette, CO, USA).

Techniques: In Vitro, Expressing, Inhibition, Incubation, Knockdown, Control, Irradiation, Comparison, Immunostaining

Journal: Oncogene

Article Title: p53 inhibits mRNA 3' processing through its interaction with the CstF/BARD1 complex.

doi: 10.1038/onc.2011.29

Figure Lengend Snippet: Figure 3 The levels of 30 cleavage inversely correlate with expression levels of p53. (a) Samples from cells expressing different levels of p53 show different levels of mRNA 30 processing. NEs from RKO, RKO-E6 and H-1299 cells were treated with UV irradiation, allowed to recover for 2 h and then were analyzed for L3 pre-mRNA 30 cleavage. NEs from RKO cells treated with p53/control siRNA and UV irradiation were also analyzed. HeLa cell samples were also included as a control. Positions of pre-mRNA and the 50 cleavage product are indicated. (b) p53 can inhibit 30 cleavage in samples from RKO-E6 and H-1299 cells. NEs from RKO-E6 and H-1299 cells were preincubated with no addition or increasing amounts of recombinant full-length His-p53 derivative (40, 80 and 120 ng). After 15 min, L3 pre-mRNA was added and incubation continued for 90 min. RNAs were purified and analyzed by denaturing PAGE. Positions of pre-mRNA and the 50 cleavage product are indicated. (c) Comparison of p53 expression levels in NE from RKO, RKO- E6, H-1299 and HeLa cells. Protein concentrations in NE from UV-treated/untreated and siRNA-treated/untreated cells were normalized by immunostaining with antibodies against Topo II.

Article Snippet: Sixty micrograms of each NE was analyzed by immunoblotting with antibodies against BARD1 (H-300, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53 (SC-126, Santa Cruz), CstF2 (generously provided by Dr Manley, Columbia University, New York, NY, USA), CstF1 (10064-2-AP, Protein Tech Group, Chicago, IL, USA) and Topoisomerase II (Topo II, SC-25330, Santa Cruz). siRNA knockdown of p53 expression in HeLa and MCF7 cells The siRNA specific for human p53 was synthesized by Qiagen (Valencia, CA, USA) and the control RNA duplex used as non-silencing siRNA was obtained from Dharmacon RNA Technologies (Lafayette, CO, USA).

Techniques: Expressing, Irradiation, Control, Recombinant, Incubation, Comparison, Immunostaining

Journal: Oncogene

Article Title: p53 inhibits mRNA 3' processing through its interaction with the CstF/BARD1 complex.

doi: 10.1038/onc.2011.29

Figure Lengend Snippet: Figure 4 The S241F mutation in p53 disrupts the BARD1, CstF and p53 interaction and decreases the inhibitory effect of p53 on 30 cleavage, and all this is restored by WT-p53 expression. (a) CstF, p53 and BARD1 do not co-immunoprecipitate from NE of DLD-1 cells that express S241F mutant p53. NEs were immunoprecipitated with either anti-CstF2 or anti-p53 antibodies. Protein samples were treated with RNase A. Equivalent amounts of the pellets (IP) and the supernatants (SN) were resolved by sodium dodecyl sulfate– polyacrylamide gel electrophoresis and proteins were detected by immunoblotting with antibodies against BARD1, CstF2 and p53. Antibodies against Topo II were used as a control of specificity. Positions of Topo II, BARD1, CstF2 and p53 are indicated. Twenty percent of the NE used in the immunoprecipitation reaction is shown as input. (b) CstF, BARD1 and p53 co-immunoprecipitate from NEs of D-A2 cells following induction of WT-p53 expression. D-A2 cells were grown in the absence of Dox to induce the expression of WT-p53. Samples were analyzed as in (a). (c) The induced expression of WT-p53 in D-A2 cells inhibits pre-mRNA 30 cleavage. NEs from DLD-1 (left panel) and D-A2 cells (right panel) non-treated or treated with Dox and/or UV irradiation, and allowed to recover for the times indicated in the figure, were analyzed for L3 pre-mRNA 30 cleavage. Positions of pre-mRNA and the 50 cleavage product are indicated. (d) The S241F mutation in p53 abolishes the inhibition of 30 cleavage. NEs from HeLa cells were preincubated with no addition or increasing amounts of recombinant His-p53 or His-p53 (S241F) derivative (40, 80 and 120 ng). After 15 min, L3 pre-mRNA was added and incubation continued for 90 min. RNAs were purified and analyzed by denaturing PAGE. Positions of pre-mRNA and the 50 cleavage product are indicated.

Article Snippet: Sixty micrograms of each NE was analyzed by immunoblotting with antibodies against BARD1 (H-300, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53 (SC-126, Santa Cruz), CstF2 (generously provided by Dr Manley, Columbia University, New York, NY, USA), CstF1 (10064-2-AP, Protein Tech Group, Chicago, IL, USA) and Topoisomerase II (Topo II, SC-25330, Santa Cruz). siRNA knockdown of p53 expression in HeLa and MCF7 cells The siRNA specific for human p53 was synthesized by Qiagen (Valencia, CA, USA) and the control RNA duplex used as non-silencing siRNA was obtained from Dharmacon RNA Technologies (Lafayette, CO, USA).

Techniques: Mutagenesis, Expressing, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Western Blot, Control, Irradiation, Inhibition, Recombinant, Incubation