Journal: Nucleic acids research

Article Title: SMG5-PNRC2 is functionally dominant compared with SMG5-SMG7 in mammalian nonsense-mediated mRNA decay.

doi: 10.1093/nar/gks1222

Figure Lengend Snippet: Figure 2. PNRC2 preferentially associates with SMG5. (A) IPs of SMG5, 6 and 7. HEK293T cells were transiently transfected with plasmid expressing FLAG, FLAG-SMG5, FLAG-SMG6 or FLAG-SMG7. Two days after transfection, cell lysates were obtained and treated with RNase A. And then IPs were performed using the a-FLAG antibody. (B) IPs of Myc-PNRC2. HEK293T cells were transiently co-transfected with the indicated plasmids. IPs were performed using the a-Myc antibody or mouse (m) IgG as a non-specific control. (C and D) IPs of FLAG-SMG5 using the extracts of cells depleted of PNRC2. (C) The selective downregulation of endogenous PNRC2 by siRNA transfection was demonstrated by western blotting. To demonstrate the quantitativity of western blotting, 3-fold serial dilutions of total-cell extracts were loaded in the three left-most lanes. (D) IPs were performed using the a-FLAG antibody or mIgG. The level of co-immunoprecipitated Dcp1a was normalized to the level of immunoprecipitated FLAG-SMG5. The normalized level of Dcp1a in the IP using the a-FLAG antibody and the undepleted cell extracts was set to 1.

Article Snippet: The following antibodies were used: FLAG (Sigma-Aldrich), HA (Roche), Myc (Calbiochem), SMG5 (Abcam), SMG6 (28), SMG7 (Bethyl), Upf1 (a gift from Lynne E. Maquat), phospho-S1078-Upf1 (21), phospho-S1096-Upf1 (21), phospho-S/TQ (Cell Signaling Technology), PNRC2 (16), Dcp1a (21) and b-actin (Sigma-Aldrich).

Techniques: Transfection, Plasmid Preparation, Expressing, Control, Western Blot, Immunoprecipitation

Journal: Nucleic acids research

Article Title: SMG5-PNRC2 is functionally dominant compared with SMG5-SMG7 in mammalian nonsense-mediated mRNA decay.

doi: 10.1093/nar/gks1222

Figure Lengend Snippet: Figure 4. Rapid mRNA degradation by tethered SMG6 requires Upf1. HeLa cells were transiently transfected with either Upf1 siRNA or non-specific Control siRNA. Two days after transfection, cells were retransfected with four plasmids: (i) a tethering reporter pcb-6bs test plasmid, (ii) an effector plasmid expressing MS2-HA or MS2-HA-SMG6, (iii) plasmid encoding siRNA-resistant (R) Myc-Upf1R either WT or T28A and (iv) the reference plasmid phCMV-MUP. (A) Western blotting showing specific downregulation by siRNAs and relative expressions of transiently expressed Myc-Upf1 WT and T28A. (B) Western blotting showing relative expressions of tethered proteins. (C) qRT–PCR of b-6bs mRNAs. The level of b-6bs mRNA was normalized to the level of MUP mRNA. Normalized levels of b-6bs mRNA obtained from cells expressing MS2-HA were set to 1.0. **P < 0.01.

Article Snippet: The following antibodies were used: FLAG (Sigma-Aldrich), HA (Roche), Myc (Calbiochem), SMG5 (Abcam), SMG6 (28), SMG7 (Bethyl), Upf1 (a gift from Lynne E. Maquat), phospho-S1078-Upf1 (21), phospho-S1096-Upf1 (21), phospho-S/TQ (Cell Signaling Technology), PNRC2 (16), Dcp1a (21) and b-actin (Sigma-Aldrich).

Techniques: Transfection, Control, Plasmid Preparation, Expressing, Western Blot, Quantitative RT-PCR

Journal: Nucleic acids research

Article Title: SMG5-PNRC2 is functionally dominant compared with SMG5-SMG7 in mammalian nonsense-mediated mRNA decay.

doi: 10.1093/nar/gks1222

Figure Lengend Snippet: Figure 6. Model illustrating the diverse combinations of Upf1-interacting factors involved in triggering NMD. Hp-Upf1 may interact with one of binding partners including SMG5-7 and PNRC2. SMG6 triggers endonucleolytic cleavage, whereas SMG5, SMG7 and PNRC2 trigger decapping activity of Dcp2 in either Dcp1a-dependent or Dcp1a-independent (unknown protein X-dependent) manner. Decapped mRNA would be vulnerable to 50-to-30-degradation. For exosome-mediated 30-to-50-decay, it is not clearly known how the hp-Upf1 activates 30-to-50-decay and what kinds of proteins are involved in this connection. The light-yellow oval indicates a functionally dominant connection shown in this study. P, phosphate group; X and Y, unidentified proteins.

Article Snippet: The following antibodies were used: FLAG (Sigma-Aldrich), HA (Roche), Myc (Calbiochem), SMG5 (Abcam), SMG6 (28), SMG7 (Bethyl), Upf1 (a gift from Lynne E. Maquat), phospho-S1078-Upf1 (21), phospho-S1096-Upf1 (21), phospho-S/TQ (Cell Signaling Technology), PNRC2 (16), Dcp1a (21) and b-actin (Sigma-Aldrich).

Techniques: Binding Assay, Activity Assay

Journal:

Article Title: Dual regulation by AP endonuclease-1 inhibits gastric epithelial cell apoptosis during Helicobacter pylori infection

doi: 10.1158/0008-5472.CAN-09-4136

Figure Lengend Snippet: Reduction of APE-1 level enhances H. pylori-mediated apoptosis via the extrinsic pathway. pSIREN and shRNA cells were treated with H. pylori (MOI 100) for 4, 8 or 12 h or left uninfected. Western analysis was performed for (A) active caspase 8 and (B) FLIPL and FLIPS. Corresponding densitometry data, normalized to α-tubulin, are depicted as the mean ± SEM of three separate experiments. *, p<0.05 for shRNA cells compared to corresponding pSIREN cells. C, caspase 8 activity was measured in AGS, pSIREN and shRNA cells with or without H. pylori infection (MOI 100) for 4, 8 or 12 h. Bars depict normalized data (mean ± SEM, n=3), *, p <0.05 for shRNA cells compared to corresponding pSIREN cells. D, immunoprecipitation of DISC with Fas antibody at 2 and 4 h after H. pylori infection in pSIREN and shRNA cells. Western analysis was performed to detect pro-caspase 8, FADD, FLIPL and FLIPS. IgG was used as the loading control. Corresponding densitometry data normalized to IgG are depicted as the mean ± SEM of three separate experiments. *, p<0.05 for shRNA cells compared to corresponding pSIREN cells.

Article Snippet: Primary antibodies that were used included APE-1 (36 kD, Novus Biologicals), cleaved caspase 3 (19 and 17 kD), cleaved PARP (89kD), cleaved caspase 8 (18kD), pro-caspase 8 (57, 43 kD), cleaved caspase 9 (37kD), cytochrome c (14 kD), FLIP L / FLIP S (58/30 kD), FADD (28 kD) (all from Cell Signaling), Bcl-x S /Bcl-x L (29/21 kD, Chemicon), Bax (21 kD, BD Pharmingen) and α-tubulin (Abcam).

Techniques: shRNA, Western Blot, Activity Assay, Infection, Immunoprecipitation, Control

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Evaluation of Eribulin Combined with Irinotecan for Treatment of Pediatric Cancer Xenografts.

doi: 10.1158/1078-0432.CCR-19-1822

Figure Lengend Snippet: Pharmacodynamic analysis of eribulin, irinotecan and the combination in ES-4 xenografts. (A) Immunoblotting of three independent ES-4 tumor lysates for markers of mitotic arrest (P-S10-histone H3), DNA damage, (P-S139-H2A.X; P-S345-Chk1; P-T68-Chk2; RRM2B, ribonucleotide reductase regulatory TP53-inducible subunit M2B), TP53-regulated genes (TP53; cyclin dependent kinase inhibitor 1A, p21; MDM2; PUMA) and apoptosis (full length and cleaved caspase 3; cleaved PARP). GAPDH was used as a loading control. (B) Immunohistochemistry for TP53 in ES-4 xenografts treated with eribulin, irinotecan or the combination. Tumors were excised and processed at 24h and 144h after the first drug dose. (C) Quantification of TP53 staining in ES-4 xenografts. *p < 0.033, **p < 0.002, ***p < 0.001 compared to control; #p < 0.033 compared to eribulin + irinotecan at 24 h; $p < 0.033, $$p < 0.002 compared to eribulin + irinotecan at 144h, two-way ANOVA with Tukey’s post-hoc test. ERB, eribulin; IRN, irinotecan; ERB+IRN, combination treatment.

Article Snippet: Proteins were transferred to Immobilon-FL PVDF membranes (MilliporeSigma) and probed with antibodies for cleaved PARP (Cell Signaling #5625), phospho-S139-H2A.X (#9718), TP53 (#48818), CDKN1A/p21 (#2947), MDM2 (#86934), PUMA/BBC3 (#12450) phospho-S345-Chk1 (#2348), Chk1 (#2360), phospho-T68-Chk2 (#2197), Chk2 (#3440), full length/cleaved caspase 3 (#9668), cleaved caspase 3 (#9664), phospho-S10-histone H3 (#53348), histone H3 (#4499), GAPDH (#2118) or RRM2B/p53R2 (Invitrogen #PA5–19970).

Techniques: Western Blot, Immunohistochemistry, Staining