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: bioRxiv

Article Title: Galectin-9 has non-apoptotic cytotoxic activity towards Acute Myeloid Leukemia independent of cytarabine resistance

doi: 10.1101/2023.01.12.523722

Figure Lengend Snippet: PS-exposure detected by flow cytometry in Gal-9-treated (300nM, 16h) (A) THP-1 cells, CD34 + patient-derived AML cells in (B) liquid, or (C) MS5 co-cultures. (D) Quantification of PS-exposure after blockade of pan-caspases with Z-VAD-FMK in Gal-9-treated THP-1 and HL-60 cells, using staurosporine (STS) as a positive control. (E) As in (D) but determined on patient-derived CD34 + and CD34 - cells. (F) Western blot of full-length caspase-3 (35 kDa) and cleaved caspase-3 (17 kDa) in HL-60 treated with Gal-9 (150, 300nM, 16h) or STS (25, 50ng/ml, 16h). Blockade of Gal-9-mediated PS-exposure with α-lactose (40mM) in (G) a panel of AML cell lines, (H) CD34 + /CD34 - patient-derived AML cells in liquid culture, or (I) in co-culture with MS5.

Article Snippet: After blocking in 5% (w/v) milk powder/TBST, proteins were detected using primary antibodies against LC3B (LC3B Antibody Kit for Autophagy, Invitrogen™, L10382, Carlsbad, CA, USA), SQSTM1/ p62 (SantaCruz, sc-28359, California, USA), β-actin (Abcam, ab49900, Cambridge, UK), total caspase 3 (#9665 cell signaling, Danvers, Massachusetts, USA), cleaved caspase 3 (#9661 cell signaling, Danvers, Massachusetts, USA), and appropriate secondary HRP-conjugated antibodies (Dako, p0217, p0260, Santa Clara, USA).

Techniques: Flow Cytometry, Derivative Assay, Positive Control, Western Blot, Co-Culture Assay

Journal: Antioxidants

Article Title: Nrf2 Activation Does Not Protect from Aldosterone-Induced Kidney Damage in Mice

doi: 10.3390/antiox12030777

Figure Lengend Snippet: Localization of the activated transcription factor Nrf2. Representative pictures of the Western blots of the expression of total Nrf2 phosphorylated at Ser40 (pNrf2, 110 kDa) in ( a ) nuclear and ( b ) cytosolic extracts of kidneys of mice as well as the quantification of band densities of the above-mentioned protein measured via ImageJ and related to the housekeepers lamin B2 (68 kDa) and GAPDH (37 kDa). Percentage of pNrf2-positive stained nuclei in cortex ( c ) and medulla ( d ). For the quantification of positive Nrf2 nuclei, 10 visual fields of cortical and 3–5 visual fields of medullary kidney sections were analyzed per animal via ImageJ. ( e ) Representative images of double stained kidney sections used for the localization of pNrf2 in kidney cells. Double staining on paraffin-embedded kidney sections was carried out using antibodies against pNrf2 (brown staining) and against calbindin (purple staining), a marker for distal tubule and early collecting duct cells. Examples of pNrf2-positive stained nuclei are indicated by black arrows; white arrows indicate the corresponding section of the tubulus system. Proximal tubules were identified by the presence of the brush border, whereas glomeruli were identified by their capillary tuft (blue circles). Distal tubules and the early collecting duct were visualized by positive calbindin staining. The late collecting duct was identified by the absence of positive calbindin staining and brush border. ( f – i ) Quantification of pNrf2-positive nuclei in the four kidney structures related to the number of nuclei in regions positive for the specific kidney cell identifiers in 10 visual fields. For the quantification in the glomerulus, 50 glomeruli were evaluated. Aldo: aldosterone, C: control, Nrf2: nuclear factor erythroid 2-related factor 2, Sulf: sulforaphane, WT: wild type. Data are shown as mean + SEM, n = 7–8. * p ≤ 0.05 vs. WT-C, # p ≤ 0.05 vs. WT-Sulf, ° p ≤ 0.05 vs. WT-Aldo, ^ p ≤ 0.05 vs. Nrf2ꜛ-C.

Article Snippet: After visualization of antibody binding, the protocol was repeated with an antibody against calbindin (1:200, #2173, Cell Signaling, Herts, UK) to identify distal tubular cells and cells of the early collecting duct.

Techniques: Western Blot, Expressing, Staining, Double Staining, Marker, Control

Journal: EBioMedicine

Article Title: NMDA receptor modulation of glutamate release in activated neutrophils

doi: 10.1016/j.ebiom.2019.08.004

Figure Lengend Snippet: GluN2B-MDAR scaffolding proteins modulate neutrophil ROS generation. (A) Histograms illustrating ROS generation (quantified by 123-dihydrorhodamine) following stimulation with E. coli and PMA-which is reduced by ZL006 (1–50 μM) in purified primary human neutrophils (n = 7). (B) E. coli -induced ROS in whole blood samples is reduced by ZL006 in a dose-dependent manner (10–50 μM; n = 7). (C) Summary data showing effect of ZL006 on PMA-induced ROS generation in neutrophils, as quantified by 123-dihydrorhodamine. Data expressed as % respective control (mean ± sem; n = 7 subjects; *p = 0.01, ANOVA). (D) shRNA knockdown of GluN1B is associated with reduced nNOS protein expression in differentiated HL60 neutrophil-like cells. (E) PMA-induced ROS production (quantified by 123-dihydrorhodamine) is attenuated in bone-marrow derived neutrophils from wild-type and homozygous PSD-95 deficient. (F) Population data for ROS in wild-type and homozygous PSD-95 deficient bone marrow derived neutrophils (mean ± sem; n = 6–7 mice/group; *p = 0.02 unpaired t -test). (G) ERK phosphorylation in primary human neutrophils is reduced by GluN2B antagonist Co 101244 following PMA-stimulation phospho-flow cytometry in CD16+ cells, human whole blood (mean ± sem; n = 5 experiments; *p < 0.05; two-way (drug × time) ANOVA with Tukey posthoc comparison). Immunoblot above graph shows similar results obtained in five separate experiments. Numbers below123-dihydrorhodamine immunoblot denote minutes after PMA stimulation (in the presence/absence of GluN2B subunit antagonist Co 101244) or phosphate-buffered saline control. (H) PMA-induced ROS production is attenuated in bone-marrow derived neutrophils from wild-type and homozygous SAP102 deficient mice. (I) Population data for wild-type and homozygous SAP-102 deficient bone marrow derived neutrophils (mean ± sem; n = 6–7 mice/group; *p = 0.04 unpaired t -test).

Article Snippet: Membranes were incubated with the following primary antibodies (from Santa Cruz Biotechnology, Heidelberg, Germany, unless otherwise stated): GluN1 (sc-9056), NE-dlg (SAP102; sc-134,400), PSD-95 (sc-28,941), ERK (1:1000; Cell Signaling Technology, Leiden; #4695), phospho-ERK (1:1000; Cell Signaling Technology, Leiden; #4370), AKT (1:1000; Cell Signaling Technology, Leiden; #9272), phospho-AKT (S473) (1:1000; Cell Signaling Technology, Leiden; #9271), NOX-2 (1:1000; Abcam, Oxford; ab129068).

Techniques: Scaffolding, Purification, shRNA, Expressing, Derivative Assay, Flow Cytometry, Western Blot