Journal: Molecular cell

Article Title: Alternative splicing decouples local from global PRC2 activity.

doi: 10.1016/j.molcel.2024.02.011

Figure Lengend Snippet: Figure 2. Suz12 exon 4 skipping promotes PRC2.1 formation and dimerization (A) Schematic depiction of SUZ12 protein highlighting the relative position and amino acid (aa) sequence encoded by exon 4. GenBank NP_056170.2 (SUZ12-L); GenBank: NP_001308136.1 (SUZ12-S). (B) PRC2.2 structure surface rendering engaging a nucleosome according to Kasinath et al.23 Region encoded by exon 4 is shown in red; the SUZ12 domains C2, ZnB, and VEFS and SUZ12 Arg 196 in cyan; histone H3 Lys 27 in black; and the RBBP4 WD propeller in gray. (C) WB showing SUZ12 protein abundance in WT#2 and Dex4 cells. Dex4 samples display only the lower band corresponding to SUZ12-S. (D) Bar plot quantifying the SUZ12-L-specific and SUZ12-S-specific tryptic peptides acquired with PRM-targeted proteomics. (E) Volcano plot of SUZ12 IP-MS in Dex4 vs WT ESCs. Significant proteins (adj. p value % 0.05, log2FC > |1.5|) are shown as red points. Data were analyzed using empirical Bayes statistics on protein-wise linear models using limma in DEP (see STAR Methods). AEBP2 and JARID2 colored as in (B). (F) Stoichiometric ratio of PRC2 core, PRC2.1 and PRC2.2 interactors between WT and Dex4 cells relative to bait (SUZ12). Points indicate individual biological replicates (n = 3 clonal cell lines); error bars, sd. Data were analyzed using PSMs from proteomicslfq. Statistics: t test. (G) IP-WB of SUZ12 in WT, Dex4, or KO ESCs.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies AEBP2 Cell Signaling Technology RRID:AB_2798398 BrdU-GFP BD Biosciences RRID:AB_400327 EPOP home-made Beringer et al., 20166 EZH2 BD Biosciences RRID:AB_2102429

Techniques: Sequencing, Quantitative Proteomics, Targeted Proteomics, Protein-Protein interactions

Journal: International Journal of Molecular Sciences

Article Title: Optimization of Biotinylated RNA or DNA Pull-Down Assays for Detection of Binding Proteins: Examples of IRP1, IRP2, HuR, AUF1, and Nrf2

doi: 10.3390/ijms24043604

Figure Lengend Snippet: The IRP-IRE pull-down assays and comparison between streptavidin-agarose and -magnetic beads. ( A ) Expression of the human ferritin H gene is regulated at least three cis-acting elements. The wild type (wt) and mutant (mt) IRE (iron responsive element) RNA sequences used for pull-down probes are shown. They were 5’-end biotinylated [Btn]. ( B ) 500 μg of SW480 WCLs (60 μL in RIPA buffer) and 2 μg (equal to ~200 pmol) of biotinylated wt IRE RNA oligonucleotide were incubated in 140 μL of binding buffer A at room temperature for 1 h with constant rotation. 0–20 μL of streptavidin magnetic beads (NEB) or 20 μL of high capacity streptavidin agarose (ThermoFisher Scientific) were washed once with washing buffer, resuspended in 50 μL of binding buffer A, added to the IRE probe/WCLs mixture, and further incubated for 1 hr. The magnetic beads were collected using a magnetic stand, and agarose resins were precipitated by micro-centrifugation at 5000 rpm for 0.5 min. They were washed twice with 1 mL of washing buffer. 12 μL of 2xSDS-PAGE sample buffer was added to precipitated resins, vortexed briefly, and heated at 95 °C for 10 min. After brief spinning, the samples were loaded on 10% SDS-PAGE gel and subjected to IRP2 western blotting. WCLs of HEK293 cells transfected with empty vector, IRP2, or IRP1 expression plasmid was loaded to verify the specificity of anti-IRP2 antibody ( B – D , F ). ( C ) lanes a–e: 500 μg of SW480 WCLs (50 μL in IP lysis buffer) and 2 μg of biotinylated wt IRE RNA oligonucleotide were incubated in 150 μL of magnetic beads binding buffer (MB, lanes a–d) or binding buffer A (lane e), followed by incubation and pull-down with 0–20 uL of pre-washed streptavidin magnetic beads (lanes a–d) or 20 μL of high capacity streptavidin agarose (lane e). lanes f, g: 500 μg of SW480 WCLs (60 μL in RIPA buffer) and 2 μg of biotinylated wt IRE RNA oligonucleotide were incubated in 140 μL of magnetic beads binding buffer (MB, lane f) or binding buffer A (lane g), pull-down with 20 μL of high capacity streptavidin agarose (lane g), and IRP2 western blotting. ( D ) 500 μg of SW480 WCLs (50 μL in IP lysis buffer) and 2 μg of biotinylated wt IRE RNA oligonucleotide were incubated in 150 μL of magnetic beads binding buffer (MB, lanes a and d), binding buffer A (lanes b, e, and g), or binding buffer C (lanes c, f, and h) followed by pull-down with 20 μL or 40 μL of pre-washed streptavidin magnetic beads (lanes a–f) or 20 μL of high capacity streptavidin agarose (lane g and h), and IRP2 western blotting. ( E ) 250 μg of K562 WCLs (20 μL in IP lysis buffer) and 2 μg of biotinylated wt IRE RNA oligonucleotide were incubated in 180 μL of binding buffer A, and pull-down with 0–30 uL of pre-washed high capacity streptavidin agarose and IRP2 western blotting. ( F ) 500 μg of SW480 WCLs (130 μL in RIPA buffer) and 2 μg of biotinylated wt or mt IRE RNA oligonucleotide were incubated in 370 μL of binding buffer A (total 500 μL) in the absence of presence of 2 μg and 8 μg of non-biotinylated wt and mt RNA oligonucleotide competitors. The binding complex was pulled down with 20 μL of pre-washed high capacity streptavidin agarose and IRP2 western blotting. All experiments were repeated 2–3 times and the representative western blots are shown.

Article Snippet: Anti-IRP1 rabbit monoclonal antibody (ab126595, lot YI071316CS, abcam, Waltham, MA, USA), 1000-fold dilution with 5% skim milk in Tween/TBS, 4 °C overnight.

Techniques: Magnetic Beads, Expressing, Mutagenesis, Incubation, Binding Assay, Centrifugation, SDS Page, Western Blot, Transfection, Plasmid Preparation, Lysis

Journal: International Journal of Molecular Sciences

Article Title: Optimization of Biotinylated RNA or DNA Pull-Down Assays for Detection of Binding Proteins: Examples of IRP1, IRP2, HuR, AUF1, and Nrf2

doi: 10.3390/ijms24043604

Figure Lengend Snippet: Verification of the IRP-IRE pull-down assay for semi-quantitative detection of IRP2 and IRP1 binding activity. ( A ) 500 μg of SW480 WCLs (30 μL in IP lysis buffer) or ( B ) K562 WCL (40 μL in IP lysis buffer), together with 0–6 μg of biotinylated wt IRE RNA oligonucleotide were incubated in 500 μL of binding buffer A or C. The binding complex was pulled down with 20 μL of pre-washed high capacity streptavidin agarose and subjected to IRP2 and IRP1 western blotting. ECL Clarity and Clarity Max (BIO-RAD) was used for IRP2 and IRP1, respectively. ( C ) 0–500 μg of K562 and SW480 WCLs were incubated with 2 μg of the biotinylated wt IRE RNA oligonucleotide in 200 μL of binding buffer A. The binding complex was pulled down with 20 μL of pre-washed high capacity streptavidin agarose and subjected to IRP2 and IRP1 western blotting. ( D ) 250 μg of WCLs (25 μL in IP lysis buffer) from K562 cells treated with 250 μM FAC or 25 μM DFO for 26 hr were incubated in 150 μL buffer C together with 4 μg of the biotinylated wt IRE probe and 20 μL of high capacity streptavidin agarose (ThermoFisher Scientific), streptavidin agarose (Invitrogen), or 30 μL of Dynabeads M-280 (Invitrogen) simultaneously. The procedures of pull-down/beads wash and western blotting for IRP2 and IRP1 are the same as other experiments. All experiments were repeated 2–3 times and the representative western blots are shown.

Article Snippet: Anti-IRP1 rabbit monoclonal antibody (ab126595, lot YI071316CS, abcam, Waltham, MA, USA), 1000-fold dilution with 5% skim milk in Tween/TBS, 4 °C overnight.

Techniques: Pull Down Assay, Binding Assay, Activity Assay, Lysis, Incubation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Optimization of Biotinylated RNA or DNA Pull-Down Assays for Detection of Binding Proteins: Examples of IRP1, IRP2, HuR, AUF1, and Nrf2

doi: 10.3390/ijms24043604

Figure Lengend Snippet: Chemicals and reagents used in this work.

Article Snippet: Anti-IRP1 rabbit monoclonal antibody (ab126595, lot YI071316CS, abcam, Waltham, MA, USA), 1000-fold dilution with 5% skim milk in Tween/TBS, 4 °C overnight.

Techniques: Magnetic Beads, Modification, Western Blot, Protease Inhibitor, Binding Assay, Lysis, Stripping Membranes, SDS Page, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Optimization of Biotinylated RNA or DNA Pull-Down Assays for Detection of Binding Proteins: Examples of IRP1, IRP2, HuR, AUF1, and Nrf2

doi: 10.3390/ijms24043604

Figure Lengend Snippet: The IRP-IRE pull-down assays and comparison between streptavidin-agarose and -magnetic beads. ( A ) Expression of the human ferritin H gene is regulated at least three cis-acting elements. The wild type (wt) and mutant (mt) IRE (iron responsive element) RNA sequences used for pull-down probes are shown. They were 5’-end biotinylated [Btn]. ( B ) 500 μg of SW480 WCLs (60 μL in RIPA buffer) and 2 μg (equal to ~200 pmol) of biotinylated wt IRE RNA oligonucleotide were incubated in 140 μL of binding buffer A at room temperature for 1 h with constant rotation. 0–20 μL of streptavidin magnetic beads (NEB) or 20 μL of high capacity streptavidin agarose (ThermoFisher Scientific) were washed once with washing buffer, resuspended in 50 μL of binding buffer A, added to the IRE probe/WCLs mixture, and further incubated for 1 hr. The magnetic beads were collected using a magnetic stand, and agarose resins were precipitated by micro-centrifugation at 5000 rpm for 0.5 min. They were washed twice with 1 mL of washing buffer. 12 μL of 2xSDS-PAGE sample buffer was added to precipitated resins, vortexed briefly, and heated at 95 °C for 10 min. After brief spinning, the samples were loaded on 10% SDS-PAGE gel and subjected to IRP2 western blotting. WCLs of HEK293 cells transfected with empty vector, IRP2, or IRP1 expression plasmid was loaded to verify the specificity of anti-IRP2 antibody ( B – D , F ). ( C ) lanes a–e: 500 μg of SW480 WCLs (50 μL in IP lysis buffer) and 2 μg of biotinylated wt IRE RNA oligonucleotide were incubated in 150 μL of magnetic beads binding buffer (MB, lanes a–d) or binding buffer A (lane e), followed by incubation and pull-down with 0–20 uL of pre-washed streptavidin magnetic beads (lanes a–d) or 20 μL of high capacity streptavidin agarose (lane e). lanes f, g: 500 μg of SW480 WCLs (60 μL in RIPA buffer) and 2 μg of biotinylated wt IRE RNA oligonucleotide were incubated in 140 μL of magnetic beads binding buffer (MB, lane f) or binding buffer A (lane g), pull-down with 20 μL of high capacity streptavidin agarose (lane g), and IRP2 western blotting. ( D ) 500 μg of SW480 WCLs (50 μL in IP lysis buffer) and 2 μg of biotinylated wt IRE RNA oligonucleotide were incubated in 150 μL of magnetic beads binding buffer (MB, lanes a and d), binding buffer A (lanes b, e, and g), or binding buffer C (lanes c, f, and h) followed by pull-down with 20 μL or 40 μL of pre-washed streptavidin magnetic beads (lanes a–f) or 20 μL of high capacity streptavidin agarose (lane g and h), and IRP2 western blotting. ( E ) 250 μg of K562 WCLs (20 μL in IP lysis buffer) and 2 μg of biotinylated wt IRE RNA oligonucleotide were incubated in 180 μL of binding buffer A, and pull-down with 0–30 uL of pre-washed high capacity streptavidin agarose and IRP2 western blotting. ( F ) 500 μg of SW480 WCLs (130 μL in RIPA buffer) and 2 μg of biotinylated wt or mt IRE RNA oligonucleotide were incubated in 370 μL of binding buffer A (total 500 μL) in the absence of presence of 2 μg and 8 μg of non-biotinylated wt and mt RNA oligonucleotide competitors. The binding complex was pulled down with 20 μL of pre-washed high capacity streptavidin agarose and IRP2 western blotting. All experiments were repeated 2–3 times and the representative western blots are shown.

Article Snippet: rabbit monoclonal anti-Aconitase 1 (IRP1) , Abcam , ab126595 , YI071316CS.

Techniques: Magnetic Beads, Expressing, Mutagenesis, Incubation, Binding Assay, Centrifugation, SDS Page, Western Blot, Transfection, Plasmid Preparation, Lysis

Journal: International Journal of Molecular Sciences

Article Title: Optimization of Biotinylated RNA or DNA Pull-Down Assays for Detection of Binding Proteins: Examples of IRP1, IRP2, HuR, AUF1, and Nrf2

doi: 10.3390/ijms24043604

Figure Lengend Snippet: Verification of the IRP-IRE pull-down assay for semi-quantitative detection of IRP2 and IRP1 binding activity. ( A ) 500 μg of SW480 WCLs (30 μL in IP lysis buffer) or ( B ) K562 WCL (40 μL in IP lysis buffer), together with 0–6 μg of biotinylated wt IRE RNA oligonucleotide were incubated in 500 μL of binding buffer A or C. The binding complex was pulled down with 20 μL of pre-washed high capacity streptavidin agarose and subjected to IRP2 and IRP1 western blotting. ECL Clarity and Clarity Max (BIO-RAD) was used for IRP2 and IRP1, respectively. ( C ) 0–500 μg of K562 and SW480 WCLs were incubated with 2 μg of the biotinylated wt IRE RNA oligonucleotide in 200 μL of binding buffer A. The binding complex was pulled down with 20 μL of pre-washed high capacity streptavidin agarose and subjected to IRP2 and IRP1 western blotting. ( D ) 250 μg of WCLs (25 μL in IP lysis buffer) from K562 cells treated with 250 μM FAC or 25 μM DFO for 26 hr were incubated in 150 μL buffer C together with 4 μg of the biotinylated wt IRE probe and 20 μL of high capacity streptavidin agarose (ThermoFisher Scientific), streptavidin agarose (Invitrogen), or 30 μL of Dynabeads M-280 (Invitrogen) simultaneously. The procedures of pull-down/beads wash and western blotting for IRP2 and IRP1 are the same as other experiments. All experiments were repeated 2–3 times and the representative western blots are shown.

Article Snippet: rabbit monoclonal anti-Aconitase 1 (IRP1) , Abcam , ab126595 , YI071316CS.

Techniques: Pull Down Assay, Binding Assay, Activity Assay, Lysis, Incubation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Optimization of Biotinylated RNA or DNA Pull-Down Assays for Detection of Binding Proteins: Examples of IRP1, IRP2, HuR, AUF1, and Nrf2

doi: 10.3390/ijms24043604

Figure Lengend Snippet: Chemicals and reagents used in this work.

Article Snippet: rabbit monoclonal anti-Aconitase 1 (IRP1) , Abcam , ab126595 , YI071316CS.

Techniques: Magnetic Beads, Modification, Western Blot, Protease Inhibitor, Binding Assay, Lysis, Stripping Membranes, SDS Page, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Microsporidia Promote Host Mitochondrial Fragmentation by Modulating DRP1 Phosphorylation

doi: 10.3390/ijms23147746

Figure Lengend Snippet: The activation of PGAM5 by E. hellem . Western blot analysis of the activation of PGAM5 in E. hellem -infected HFF ( A ) and HEK293 ( B ) cells at 48 hpi, respectively. GAPDH was used as an internal control. Red arrows mark the cleaved PGAM5 bands with significant changes. The relative optical density of cleaved PGAM5 bands was determined and statistically compared. ( C ) Transcript level of PGAM5 . GAPDH used as an internal control gene. Mock, no treatment; NC, Negative control. ( D ) Western blot analysis of the dephosphorylation and phosphorylation of DRP1 with knockdown PGAM5 at 36 h. Simultaneously, the light-observed HEK293 cells are shown in the lower panels ( E ). Arrows indicate the PV of E. hellem . Bar, 50 μm. ( F ) The relative optical density of cleaved PGAM5 immune blots. ( G ) Statistical analysis of the relative optical density of immune blots for DRP1 Ser616. ( H ) Statistical analysis of the relative optical density of immune blots for DRP1 Ser637. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Anti-TOM20 rabbit monoclonal antibody (ab186735, Abcam, Cambridge, UK), anti-DRP1 mouse monoclonal (sc-101270, Santa Cruz, Dallas, TX, USA), anti-DRP1 (phospho Ser637) rabbit polyclonal (ab193216, Abcam), anti-DRP1 (phospho Ser616) rabbit polyclonal (#3455S, Cell Signalling Technology, MA, USA), anti-OPA1 mouse monoclonal (sc-393296, Santa Cruz), anti-MFN1 mouse monoclonal (sc-166644, Santa Cruz), anti-MFN2 mouse monoclonal (sc-515647, Santa Cruz), anti-GAPDH rabbit monoclonal (AF1186, Beyotime, Nantong, China), anti-β Tubulin mouse monoclonal (sc-166729, Santa Cruz), anti-COX4 rabbit monoclonal (ab202554, Abcam), anti-FIS1 rabbit monoclonal (ab156865, Abcam), anti-PGAM5 mouse monoclonal (sc-515880, Santa Cruz), and anti-PGAM5 rabbit monoclonal (ab126534, Abcam) were used.

Techniques: Activation Assay, Western Blot, Infection, Negative Control, De-Phosphorylation Assay

Journal: International Journal of Molecular Sciences

Article Title: Microsporidia Promote Host Mitochondrial Fragmentation by Modulating DRP1 Phosphorylation

doi: 10.3390/ijms23147746

Figure Lengend Snippet: A model for microsporidia modulating HMF. Microsporidian infection promotes HMF by activating PGAM5, which dephosphorylates DRP1 Ser637. Moreover, the infection also increases the phosphorylation of DRP1 Ser616. Both modifications result in the translocation of DRP1 from cytosol onto the mitochondrial outer membrane, and mediates the fragmentation of mitochondria. The significant increase in HMF would up-regulate host metabolism and provide energy and nutrients for the proliferation of microsporidia. PGAM5, phosphoglycerate mutase 5; DRP1, dynamin 1-like protein.

Article Snippet: Anti-TOM20 rabbit monoclonal antibody (ab186735, Abcam, Cambridge, UK), anti-DRP1 mouse monoclonal (sc-101270, Santa Cruz, Dallas, TX, USA), anti-DRP1 (phospho Ser637) rabbit polyclonal (ab193216, Abcam), anti-DRP1 (phospho Ser616) rabbit polyclonal (#3455S, Cell Signalling Technology, MA, USA), anti-OPA1 mouse monoclonal (sc-393296, Santa Cruz), anti-MFN1 mouse monoclonal (sc-166644, Santa Cruz), anti-MFN2 mouse monoclonal (sc-515647, Santa Cruz), anti-GAPDH rabbit monoclonal (AF1186, Beyotime, Nantong, China), anti-β Tubulin mouse monoclonal (sc-166729, Santa Cruz), anti-COX4 rabbit monoclonal (ab202554, Abcam), anti-FIS1 rabbit monoclonal (ab156865, Abcam), anti-PGAM5 mouse monoclonal (sc-515880, Santa Cruz), and anti-PGAM5 rabbit monoclonal (ab126534, Abcam) were used.

Techniques: Infection, Translocation Assay

Journal: Cells

Article Title: Enhanced Cognition and Neurogenesis in miR-146b Deficient Mice.

doi: 10.3390/cells11132002

Figure Lengend Snippet: Figure 5. Increased adult hippocampal neurogenesis in Mir146b-/- mice. (A) Representative immuno- histochemistry microphotographs of the hippocampal Ki67+ cells at 100× magnification and inserted microphotographs at 1000× magnification. (B) Illustrative microphotographs of the hippocam- pal BrdU+ cells at 100× magnification and inserted microphotographs at 1000× magnification. (C) Represented microphotographs of doublecortin positive cells taken at 100× magnification. (D) Illustrative images of BrdU, GFAP and calbindin signal and their co-localization in the hip- pocampus. (E) Quantitative graph showing increased number of Ki67 positive cells. (F) Increased number of BrdU+ cells. (G) Quantitative graphs showing number of doublecortin positive cells in the hippocampus of WT and Mir146b-/- mice. (H) Percentage of BrdU+ cells with GFAP and (I) calbindin in the dentate gyrus of WT and Mir146b-/- mice. Scale bar = 20 µm. Number of animals = 6, Student’s t-test. Data represented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For immunofluorescent double-labelling, sections were incubated with a mixture of anti-BrdU monoclonal antibody (1:200, RF04-2, Bio-Rad, Hercules, CA, USA) and rabbit anti-calbindin antibody (1:800, AB1778, Chemicon International Inc, Temecula, CA, USA) or rabbit anti-GFAP (1:800, Z0334, Dako, Glostrup, Denmark).

Techniques: Immunohistochemistry