Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Diet-induced aortic valve disease in mice haploinsufficient for the Notch pathway effector RBPJK/CSL.

doi: 10.1161/ATVBAHA.111.227561

Figure Lengend Snippet: Figure 2. Greater leaflet thickness and increased macrophage deposition in RBPKO/ mice fed an HCVD diet. Shown are Masson Trichrome staining (A to F) of aortic valves indicating collagen deposition (arrows) and Mac3 immuno- staining (G to L) of aortic valves demon- strating macrophage infiltration (red; arrows indicate positive immunostaining) in WT mice fed the CC diet (A and G) or the HCVD diet (D and J), Notch1KO/ mice fed the CC diet (B and H) or the HCVD diet (E and K), and RBPKO/ mice fed the CC diet (C and I) or the HCVD diet (F and L). M and N, Mean data of cusp area (M) and percentage of positively stained sections for Mac3 (N) for all groups (n4 per group). One-way ANOVA and LSD post hoc analysis were used to compare differences between groups. Bars with different letters indi- cate significant differences (P0.05) vs all other groups (LSD post hoc analysis). Scale bar0.1 mm.

Article Snippet: Inflammation was detected with anti-Mac3 rat monoclonal antibody (SC19991, Santa Cruz Technol, 1:200).

Techniques: Staining, Immunostaining

Journal: PLoS ONE

Article Title: Rab39a Interacts with Phosphatidylinositol 3-Kinase and Negatively Regulates Autophagy Induced by Lipopolysaccharide Stimulation in Macrophages

doi: 10.1371/journal.pone.0083324

Figure Lengend Snippet: (A) Immunostaining of Raw264.7 macrophages expressing EGFP-Rab39a with anti-LAMP2 antibody. (B) Representative sequence images from FRAP analysis of EGFP-Rab39a on latex bead-containing phagosomes. The region marked by a broken-line circle was photobleached at 4 sec, and the recovery of fluorescence was monitored. (C) Temporal changes in fluorescence intensities on the bleached phagosomes. The relative intensity was defined as the ratio of fluorescence intensity at each time point to that at 0 sec. Data represent means and standard errors of means (n=10).

Article Snippet: Rat anti-mouse LAMP2 monoclonal antibody (SouthernBiotech), rabbit anti-LC3 polyclonal antibody (MBL), mouse anti-tubulin monoclonal antibody (Sigma-Aldrich), rabbit anti-p62 antibody (MBL), mouse anti-ubiquitin antibody (MBL), rabbit anti-Beclin1 antibody (MBL), rabbit anti-GM130 antibody (MBL), mouse anti-EGFP monoclonal antibody (Clonetech), rat anti-EGFP monoclonal antibody (Nacalai tesque) , mouse anti-c-Myc antibody (Wako), rabbit anti-Vps34 antibody (CST), mouse anti-UVRAG antibody (MBL) and rabbit anti-ATG14L antibody (MBL) were used as primary antibodies.

Techniques: Immunostaining, Expressing, Sequencing, Fluorescence

Journal:

Article Title: Biogenesis of Leishmania major -Harboring Vacuoles in Murine Dendritic Cells

doi: 10.1128/IAI.74.2.1305-1312.2006

Figure Lengend Snippet: Biogenesis of PV in immature BMDC (A) and FSDC (B) infected with CMFDA- or CMTMR-labeled L. major promastigotes. At 0.5, 1, 4, or 24 h after pulse infection, the cells were fixed, permeabilized, and stained with antibodies and appropriate secondary reagents to examine the association of CD71, CD68, Rab7, LAMP-1, or LAMP-2 with L. major-containing PV by confocal fluorescence microscopy. Freshly prepared BMDC were identified by CD11c labeling. For each time point and each molecule, the percentages of parasite-containing compartments that colocalized with endosomal/lysosomal proteins were determined after 50 to 300 PV were counted. The data are mean values ± standard errors of the means for two to six independent experiments.

Article Snippet: Thereafter, DC were incubated for 45 min at room temperature in a humid chamber with one of the following primary antibodies in saponin-containing blocking solution: monoclonal antibody (MAb) C2, a rat immunoglobulin G1 (IgG1) recognizing mouse CD71 (transferrin receptor) (BD Pharmingen, Heidelberg, Germany), fluorescein isothiocyanate-conjugated rat anti-mouse LAMP-2 MAb (BD Pharmingen), rabbit anti-mouse Rab4 antibodies (Santa Cruz, Santa Cruz, Calif.), rabbit anti-mouse Rab7 antibodies (Santa Cruz), phycoerythrin-conjugated rat anti-mouse LAMP-1 MAb (Santa Cruz), and biotin-conjugated rat anti-mouse CD68 (macrosialin) MAb (Serotec, Oxford, United Kingdom).

Techniques: Infection, Labeling, Staining, Fluorescence, Microscopy

Journal:

Article Title: Biogenesis of Leishmania major -Harboring Vacuoles in Murine Dendritic Cells

doi: 10.1128/IAI.74.2.1305-1312.2006

Figure Lengend Snippet: Biogenesis of PV in immature BMDC (A) and mature BMDC (B) infected with CMFDA- or CMTMR-labeled L. major promastigotes. At 0.5, 1, 2, or 4 h after pulse infection, the cells were fixed, permeabilized, and stained with antibodies and appropriate secondary reagents to examine the association of CD68, LAMP-1, LAMP-2, CD71, Rab7, or Rab4 with L. major-containing PV by confocal fluorescence microscopy. DC were identified by CD11c labeling. For each time point and each molecule, the percentages of parasite-containing compartments that colocalized with endosomal/lysosomal proteins were determined after 100 to 200 PV were counted. The data are mean values ± standard deviations for two independent experiments.

Article Snippet: Thereafter, DC were incubated for 45 min at room temperature in a humid chamber with one of the following primary antibodies in saponin-containing blocking solution: monoclonal antibody (MAb) C2, a rat immunoglobulin G1 (IgG1) recognizing mouse CD71 (transferrin receptor) (BD Pharmingen, Heidelberg, Germany), fluorescein isothiocyanate-conjugated rat anti-mouse LAMP-2 MAb (BD Pharmingen), rabbit anti-mouse Rab4 antibodies (Santa Cruz, Santa Cruz, Calif.), rabbit anti-mouse Rab7 antibodies (Santa Cruz), phycoerythrin-conjugated rat anti-mouse LAMP-1 MAb (Santa Cruz), and biotin-conjugated rat anti-mouse CD68 (macrosialin) MAb (Serotec, Oxford, United Kingdom).

Techniques: Infection, Labeling, Staining, Fluorescence, Microscopy

Journal: Cell Reports Medicine

Article Title: Targeting phenylpyruvate restrains excessive NLRP3 inflammasome activation and pathological inflammation in diabetic wound healing

doi: 10.1016/j.xcrm.2023.101129

Figure Lengend Snippet: Phenylpyruvate upregulated NLRP3 palmitoylation by binding to the PPT1 protein (A) Schematic view of NLRP3 (full length) and the location of the S-palmitoylation sites. (B) ABE assay and immunoblot analysis were used to evaluate NLRP3 palmitoylation in BMDMs treated with or without palmitic acid (n = 3). (C) Immunoblot and statistical analysis showing NLRP3 expression in BMDMs treated with 2BP at the indicated concentrations (0, 50, 100, and 150 μM) for 24 h (n = 3). (D) Immunoblot and statistical analysis showing NLRP3 expression in BMDMs treated with 2BP (100 μM) for 0, 8, 16, and 24 h (n = 3). (E) Immunostaining of the location of LAMP2 (indicating lysosomes), PPT1, and NLRP3 in macrophages. The nuclei were stained with Hoechst dye. Scale bar, 10 μm. (F and G) Immunoprecipitation (IP) and immunoblot analysis were used to detect the endogenous NLRP3 and PPT1 association in macrophages (n = 3). (H) ABE assay and immunoblot analysis were used to evaluate NLRP3 palmitoylation in BMDMs with Ppt1 knockdown (n = 3). (I) Mouse embryo fibroblasts (MEFs) were transfected with WT NLRP3 or the indicated NLRP3 mutants for 24 h with or without knockdown of Ppt1. ABE assay and immunoblot analysis showing palmitoylation levels of the indicated NLRP3 mutants (n = 3). (J) ABE assay and immunoblot analysis were used to determine NLRP3 palmitoylation levels in BMDMs treated with increasing phenylpyruvate concentrations (n = 3). (K) Determination of NLRP3 palmitoylation levels in BMDMs treated with 400 μM phenylpyruvate and then transfected with the Ppt1 WT plasmid or the MUT plasmid for 24 h (n = 3). (L) BMDMs were treated with 400 μM phenylpyruvate with or without 100 μM 2BP and then transfected with or without the Ppt1 MUT plasmid. ABE assay and immunoblot analysis determining NLRP3 palmitoylation levels in the indicated cells (n = 3). Data are shown as mean ± SD. ∗p < 0.05, ∗∗p < 0.01; n.s., not significant.

Article Snippet: Rat anti-mouse LAMP2 , Proteintech , Cat# 65052-1-Ig; RRID: AB_2881468.

Techniques: Binding Assay, Western Blot, Expressing, Immunostaining, Staining, Immunoprecipitation, Knockdown, Transfection, Plasmid Preparation

Journal: Cell Reports Medicine

Article Title: Targeting phenylpyruvate restrains excessive NLRP3 inflammasome activation and pathological inflammation in diabetic wound healing

doi: 10.1016/j.xcrm.2023.101129

Figure Lengend Snippet:

Article Snippet: Rat anti-mouse LAMP2 , Proteintech , Cat# 65052-1-Ig; RRID: AB_2881468.

Techniques: Recombinant, Transfection, Lysis, cDNA Synthesis, Real-time Polymerase Chain Reaction, BIA-KA, Enzyme-linked Immunosorbent Assay, Software

Journal: Nature communications

Article Title: IRF8 directs stress induced autophagy in macrophages and promotes clearance of Listeria monocytogenes

doi: 10.1038/ncomms7379

Figure Lengend Snippet: (a) Immunostaining of LAMP2 in WT and Irf8 -/- MΦs stimulated with IFNγ overnight and TLR ligands for 8 h. Cells were counterstained for DNA (blue). The scale bar: 20 μm. (b) Immunoblot detection of LAMP2 in WT and Irf8 -/- MΦs. Ten microgram of extracts was tested with antibody against LAMP2 or β-Tubulin. Below: The amounts of LAMP2 in three independent experiments were quantified using the ImageJ software. **p-value ≤0.01 (c) WT and Irf8 -/- MΦs were immunostained for LC3 antibody and LysoTracker red to detect LC3-positive and Lysosome-positive structures. Below: Cells with double positive vesicles were quantified as in . Data represents the average of three independent experiments. p-value ≤0.01 (**).The scale bar: 20 μm.

Article Snippet: Cells were incubated for 5 h with antibody against LC3 (MBL, cat. no. M152-3) or LAMP2 (ProSci, cat. no. 3627), SQSTM1 (MBL, cat. no.PM045), Ubiquitin (Santacruz, cat. no. sc8017), or Listeria (Abcam, cat. no. ab35132) diluted at 1:1000 followed by 1 h incubation with goat Alexa-488/Alexa-592 anti-rabbit or anti-mouse antibody (Molecular Probes).

Techniques: Immunostaining, Western Blot, Software