Journal: PLoS ONE

Article Title: Involvement of Girdin in the Determination of Cell Polarity during Cell Migration

doi: 10.1371/journal.pone.0036681

Figure Lengend Snippet: A. Fragments of the Par-3 4N domain used in the study are shown. The 4N domain of Par-3 was further divided into three subdomains (4N/1, 4N/2, and 4N/3). The corresponding amino acid numbers for the fragments are indicated in parenthesis. B. HEK293T cells were transfected with the indicated combination of each subdomain of myc-Par-3 4N, GST, and GST-CT. Lysates were precipitated with glutathione-Sepharose beads and eluted proteins were analysed with the indicated antibodies. Par-3 4N and 4N/2 domains that bound to GST-CT are indicated by asterisks. C. Direct interaction between the 4N/2 region of Par-3 and Girdin. Purified recombinant MBP (maltose binding protein)-fusion proteins containing the 4N, 4N/2, and 4N/1 regions of Par-3 (300 pmol) were incubated with 50 pmol of recombinant GST of GST-CT conjugated with glutathione beads for 1 hr at 4°C, washed three times, eluted with 10 mM reduced glutathione, separated on SDS-polyacrylamide gels, and subjected to Western blot analyses using the indicated antibodies. Asterisks, bound MBP-fusion proteins. White asterisks, MBP-fusion protein input. D. Total cell lysates from HEK293T cells that expressed Girdin-V5 and either myc-GST, myc-Par-3, or myc-Par-3 Δ4N/2 were immunoprecipitated with anti-myc antibody, followed by Western blot analyses using the indicated antibodies. myc-Par-3 (wild type and Δ4N/2) and Girdin-V5 are indicated by asterisks and stars, respectively.

Article Snippet: Other antibodies used in this study included anti-Par-3 rabbit polyclonal antibody (Millipore, Bedford, MA), anti-Girdin sheep polyclonal antibody (R&D Systems, Minneapolis, MN), anti-GM130 rabbit monoclonal antibody (Abcam, Cambridge, UK), anti-GM130 mouse monoclonal antibody (BD Bioscience, San Jose, CA), anti-E-cadherin mouse monoclonal antibody (BD Bioscience), anti-GST monoclonal antibody (Cell Signaling Technology, Danvers, MA), anti-MBP antiserum (New England Biolabs, Beverly, MA), anti-GFP polyclonal antibody (MBL, Nagoya, Japan), anti-β-actin mouse monoclonal antibody (Sigma, St Louis, MO), anti-γ-tubulin mouse monoclonal antibody (Sigma), anti-Dcx (doublecortin) goat polyclonal antibody (Santa Cruz Biotechnology), anti-V5 monoclonal antibody (Invitrogen, Carlsbad, CA), and anti-c-myc monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Transfection, Purification, Recombinant, Binding Assay, Incubation, Western Blot, Immunoprecipitation

Journal: International immunopharmacology

Article Title: LXA4 protects against hypoxic-ischemic damage in neonatal rats by reducing the inflammatory response via the IκB/NF-κB pathway.

doi: 10.1016/j.intimp.2020.107095

Figure Lengend Snippet: Fig. 5. Long-term neuroprotective effect of LXA4 treatment. (A) The brains were isolated from each group 7 d post HI. (B) Protein expression level of MAP-2 and MBP 24 h after HI brain injury 7 d post-HI. (C, D) Analyses of MAP-2and MBP (of β-actin). ****P < 0.0001 versus the Sham + NS group, #P < 0.05, ###P < 0.001 versus the HI + NS group. n = 3. (E) Representative images of immunohistochemical staining for microtubule-associated protein 2 (MAP-2) and myelin basic protein (MBP). The below columns show magnified images of the black boxes in the above column. Scale bar = 100 μm.

Article Snippet: The sections were incubated with the following antibodies overnight at 4 °C: MBP (1:200, Sc-13914, Santa Cruz Biotechnology) and MAP-2 (1:200, Sc-20172, Santa Cruz Biotechnology).

Techniques: Isolation, Expressing, Hi-C, Immunohistochemical staining, Staining

Journal: Cell death & disease

Article Title: Peptidylarginine deiminase 2 plays a key role in osteogenesis by enhancing RUNX2 stability through citrullination.

doi: 10.1038/s41419-023-06101-7

Figure Lengend Snippet: Fig. 4 PADI2 protects RUNX2 from ubiquitin-proteasomal degradation pathway. A–C MC3T3-E1 cells were transfected with Strep-Runx2 with or without Flag-PADI2, and then cultivated in osteogenic medium for 3 days. On day 3, 4 μg/mL Actinomycin D was treated and incubated for 0, 3, and 6 h. The half-life of Runx2 mRNA and RUNX2 protein was determined by RT-qPCR (A) and western blot analysis (B), respectively. The intensities of Strep-RUNX2 protein levels were normalized against each GAPDH by ImageJ. The normalized values at 0 h were set as 1, and relative levels are shown (C). D, E MC3T3-E1 cells were transfected with Strep-Runx2 together with or without Flag-PADI2, and then cultivated in osteogenic medium for 3 days. 20 μg/mL cycloheximide was treated on the last day and incubated for 0, 3, and 6 h. The half-life of RUNX2 protein was determined by western blot analysis (D). The intensities of Strep-RUNX2 protein levels were normalized against each GAPDH by ImageJ and relative levels are shown (E). F Padi2 control and KO pOB cells were cultured in osteogenic media for 2 days, and 20 μM MG132 or DMSO as vehicle was treated for 6 h before harvesting cells, and western blot analysis followed. β-Actin was used as a loading control. RUNX2 level was quantified using ImageJ software and normalized with β-Actin. The red arrow indicates PADI2. G MC3T3-E1 cells were transfected with siCont or siPadi2 #2 and then cultivated in osteogenic media for an additional 2 days. 20 μM MG132 or DMSO as the vehicle was treated for 6 h before harvesting cells followed by western blot analysis. α-Tubulin was used as a loading control. RUNX2 level was quantified using ImageJ software and normalized with α-Tubulin. The red arrow indicates PADI2. H hMSCs were transfected with siCont or siPADI2 and then cultivated in osteogenic media for an additional 2 days. 20 μM MG132 or DMSO as the vehicle was treated for 6 h before harvesting cells followed by western blot analysis. β-Actin was used as a loading control. RUNX2 level was quantified using ImageJ software and normalized with β-Actin. The red arrow indicates PADI2. Western blot data were collected from at least two or three independent experiments; the representative results are shown here. I Strep-Runx2, Flag-PADI2, and HA-ubiquitin were transfected into 293 T cells. 3 days after transfection, cells were treated with 20 μM MG132 for 6 h, lysed, immunoprecipitated with Strep-Tag II magnetic beads, and immunoblotted with anti-HA or anti-RUNX2 antibody. Ubiquination assay was performed in three independent experiments; the representative results are shown here.

Article Snippet: Primary antibodies to the following antigens were used: PADI2 (Proteintech), RUNX2 (MBL Life science), and Collagen type I (COL1) (sc-59772; Santa Cruz Biotechnology, Inc).

Techniques: Ubiquitin Proteomics, Transfection, Incubation, Quantitative RT-PCR, Western Blot, Control, Cell Culture, Software, Immunoprecipitation, Strep-tag, Magnetic Beads

Journal: Cell death & disease

Article Title: Peptidylarginine deiminase 2 plays a key role in osteogenesis by enhancing RUNX2 stability through citrullination.

doi: 10.1038/s41419-023-06101-7

Figure Lengend Snippet: Fig. 5 PADI2 citrullinates RUNX2 via physical interaction. A 293 T cells were transfected with 3xHA-Runx2 together with or without Flag- PADI2 and cultured for 3 days after the transfection. Cells were lysed, immunoprecipitated with anti-HA antibody and protein G-conjugated magnetic beads, and immunoblotted with indicated antibodies. The red arrow indicates predicted citrullinated RUNX2. Co-IP experiment was performed in three independent experiments; the representative results are shown here. B Workflow showing the detection of citrullinated RUNX2 by Biotin-PG labeling (left). Recombinant human RUNX2 (rhRUNX2) isoform c (NP_004339) was in vitro citrullinated by rhPADI2. The samples were labeled with Biotin-PG and then separated by SDS-PAGE followed by transfer to PVDF membrane. The membrane was incubated with Streptavidin conjugated with horseradish peroxidase (Streptavidin-HRP) or was immunoblotted with anti-RUNX2 antibody (right). A red asterisk indicates citrullinated RUNX2. This experiment was performed in three independent experiments; the representative results are shown here. C In vitro citrullinated rhRUNX2 isoform c was analyzed by LC-MS/MS. The ten arginine (R) sites of rhRUNX2 citrullinated by PADI2 were identified (left column). The mouse RUNX2 isoform 1 was used for site-directed mutagenesis of the 10 R sites and, for this, the R site in mouse RUNX2 isoform 1 matching the corresponding each R site in hRUNX2 isoform c is shown in the right column.

Article Snippet: Primary antibodies to the following antigens were used: PADI2 (Proteintech), RUNX2 (MBL Life science), and Collagen type I (COL1) (sc-59772; Santa Cruz Biotechnology, Inc).

Techniques: Transfection, Cell Culture, Immunoprecipitation, Magnetic Beads, Co-Immunoprecipitation Assay, Labeling, Recombinant, In Vitro, SDS Page, Membrane, Incubation, Liquid Chromatography with Mass Spectroscopy, Mutagenesis

Journal: Cell death & disease

Article Title: Peptidylarginine deiminase 2 plays a key role in osteogenesis by enhancing RUNX2 stability through citrullination.

doi: 10.1038/s41419-023-06101-7

Figure Lengend Snippet: Fig. 6 PADI2-catalyzed citrullination of RUNX2 is required for RUNX2 stabilization. A MC3T3-E1 cells were transfected with empty vector (EV), Strep-Runx2 wild type (Wt), or R-to-K mutants and cultured in osteogenic media for 3 days after transfection. Cells were lysed and western blot analysis was performed. β-Actin was used as a loading control. Strep-RUNX2 level was quantified using ImageJ software and normalized with β-Actin. The arrow indicates non-specific bands (n.s). Western blot data were collected from three independent experiments; the representative results are shown here. B, C pOB cells and hMSCs were transfected with EV, Strep-Runx2 WT, or R381K mutant plasmids and cultured in osteogenic media for 2 days. Cells were lysed and western blot analysis was performed. β-Actin was used as a loading control. Strep-RUNX2 level was quantified and normalized with β-Actin using ImageJ software. Western blot data were collected from two independent experiments; the representative results are shown here. D 293 T cells were transfected with Strep-Runx2 Wt or R-to-K mutants with or without Myc-Cbfβ plasmids and then incubated for 3 days after transfection. Cells were lysed, immunoprecipitated with Strep-Tag II magnetic beads, and immunoblotted with indicated antibodies. Co-IP experiment was performed in three independent experiments; the representative results are shown here. E MC3T3-E1 cells were transfected with Strep-Runx2 Wt or R-to-K mutants and cultured for 2 days after transfection. Cells were fixed with 4% PFA, permeabilized, and then immunofluorescent staining was performed using anti-Strep-Tag II antibody. DAPI was used for the nucleus. Three independent experiments were performed and the representative results are shown here. Scale bar, 20 μm.

Article Snippet: Primary antibodies to the following antigens were used: PADI2 (Proteintech), RUNX2 (MBL Life science), and Collagen type I (COL1) (sc-59772; Santa Cruz Biotechnology, Inc).

Techniques: Transfection, Plasmid Preparation, Cell Culture, Western Blot, Control, Software, Mutagenesis, Incubation, Immunoprecipitation, Strep-tag, Magnetic Beads, Co-Immunoprecipitation Assay, Staining