Journal: International Journal of Molecular Sciences

Article Title: αvβ3 Integrin as a Link between the Development of Fibrosis and Thyroid Hormones in Systemic Sclerosis

doi: 10.3390/ijms24108927

Figure Lengend Snippet: List of antibodies.

Article Snippet: Integrin β3 , Rabbit , Polyclonal Ab , Santa Cruz Biotechnology , SC14009 , 1:200 , BSA 5%.

Techniques: Hybridization

Journal: The Journal of cell biology

Article Title: Proteolytic regulation of a galectin-3/Lrp1 axis controls osteoclast-mediated bone resorption.

doi: 10.1083/jcb.202206121

Figure Lengend Snippet: Figure 4. Mmp9/Mmp14 regulates galectin-3 lattice remodeling in osteoclasts. (A and B) Galectin-3 expression and cleavage as assessed with anti–galectin-3 monoclonal antibody (ab2785; Abcam; epitopes mapped against N-terminal region) by Western blot (A) and quantification (B) in BMDMs and osteoclasts (OC) generated from wild-type or DKO mice. Data are presented as mean ± SEM (n = 3 biological replicates). (C and D) Galectin-3 (green) im- munofluorescence of non-permeabilized wild-type or DKO osteoclasts stained with an anti–galectin-3 monoclonal antibody (#125401; Biolegend; clone M3/38, epitopes mapped against N-terminal region; C), and surface galectin-3 level quantified (D). Scale bar, 20 μm. Data are presented as mean ± SEM (n = 3 biological replicates with two technical replicates each). (E) Following cell surface biotinylation and capture with streptavidin magnetic beads, Mmp9, Mmp14, galectin-3, and β3 integrin expression in the membrane fraction of wild-type and DKO osteoclasts as assessed by Western blot. Results are representative of three independent experiments. (F) Measurements of surface galectin-3 in wild-type and DKO osteoclasts with eFluor 660–conjugated anti–galectin-3 monoclonal antibody (#50-5301-82; Thermo Fisher Scientific; clone M3/38, epitopes mapped within the N-terminal domain) by flow cytometry. Results are representative of three independent experiments. (G) Measurements of surface galectin-1 in wild-type and DKO osteoclasts by flow cytometry. Results are representative of three independent experiments. (H) DKO BMDMs were transduced with lentiviral vectors expressing full-length MMP9, an MMP9E/A mutant, or an empty control, and differentiated into osteoclasts. Cell lysates were collected for MMP9 and galectin-3 expression and cleavage as assessed by Western blot. Results are representative of three independent experiments. (I) DKO BMDMs were transduced with lentiviral vectors expressing full-length MMP14, MMP14E/A, or an empty control and differentiated into osteoclasts. Cell lysates were collected for MMP14 and galectin-3 expression and cleavage as assessed by Western blot. Results are representative of three independent experiments. **P < 0.01. Statistical significance was assessed using two-way ANOVA with Bonferroni cor- rection (B) and unpaired two-sided Student’s t test (D). Source data are available for this figure: SourceData F4.

Article Snippet: Antigen detection was performed using antibodies directed against c-Src (rabbit antimouse/human antibody; #2109; Cell Signaling), Ctsk (mouse anti-mouse/human antibody; sc-48353; Santa Cruz), Rho (mouse anti-mouse/human antibody; #05-778; Millipore), galectin-3 (mouse anti-mouse/human antibody; ab2785; Abcam; epitopes mapped within the N-terminal region), Lrp1 (mouse anti-mouse antibody; MABN1796; Millipore), Mmp9 (rabbit anti-mouse antibody; ab38898; Abcam), Mmp14 (rabbit anti-mouse antibody; ab53712; Abcam), OXPHOS (rabbit anti-mouse antibody; ab110413; Abcam), vinculin (mouse anti-mouse antibody; V9131; Sigma-Aldrich), β3 integrin (rabbit anti-mouse antibody; #4702; Cell Signaling), or β-actin (rabbit anti-mouse antibody; #4970; Cell Signaling).

Techniques: Expressing, Western Blot, Generated, Staining, Magnetic Beads, Membrane, Flow Cytometry, Transduction, Mutagenesis, Control

Journal: The Journal of cell biology

Article Title: Proteolytic regulation of a galectin-3/Lrp1 axis controls osteoclast-mediated bone resorption.

doi: 10.1083/jcb.202206121

Figure Lengend Snippet: Figure 7. Mmp9/Mmp14 co-dependent regulation of osteoclast function through the galectin-3–centric control of Lrp1 activation. (A–C) Pre- osteoclasts were incubated with either human GALECTIN-3 or GALECTIN-3-biotin (hGAL3-biotin; 1.5 µM) for 30 min at 4°C. Biotinylated GALECTIN-3 was precipitated with streptavidin magnetic beads, separated by SDS-PAGE (A), and analyzed by mass spectrometry (B). Hits were classified according to the peptides spectral matches and the number of peptides per protein. Red columns: the top hit of GALECTIN-3 binding partner. The numbering on the x axis relates to Table S3. Pie chart depicts GALECTIN-3 interactors based on the GO annotation “cellular component” in FunRich (C). (D) Lrp1 and galectin-3 ex- pression from the above streptavidin magnetic beads–precipitated lysates as assessed by Western blot. Results are representative of three independent experiments. (E) Protein lysates from wild-type osteoclasts were immunoprecipitated with the galectin-3 antibody, and Lrp1 and galectin-3 levels assessed by Western blot. Results are representative of three independent experiments. (F and G) Cell-free binding assay demonstrating direct binding between 0.1 and 3.2 μg GALECTIN-3 and 0.5 μg LRP1 for 4 h at 30°C (F), and the competitive inhibition of binding between GALECTIN-3 and LRP1 (1.6 versus 0.5 μg) by 50 mM sucrose, 50 mM lactose, or 10 mg/ml GCS-100 (G). Data are presented as mean ± SEM (n = 3 biological replicates). (H) Following cell surface biotinylation and capture with streptavidin magnetic beads, Lrp1 and β3 integrin expression in the membrane fraction of wild-type and DKO osteoclasts as assessed by Western blot. Results are representative of three independent experiments. **P < 0.01. Statistical significance was assessed using one-way ANOVA with Bonferroni correction. Source data are available for this figure: SourceData F7.

Article Snippet: Antigen detection was performed using antibodies directed against c-Src (rabbit antimouse/human antibody; #2109; Cell Signaling), Ctsk (mouse anti-mouse/human antibody; sc-48353; Santa Cruz), Rho (mouse anti-mouse/human antibody; #05-778; Millipore), galectin-3 (mouse anti-mouse/human antibody; ab2785; Abcam; epitopes mapped within the N-terminal region), Lrp1 (mouse anti-mouse antibody; MABN1796; Millipore), Mmp9 (rabbit anti-mouse antibody; ab38898; Abcam), Mmp14 (rabbit anti-mouse antibody; ab53712; Abcam), OXPHOS (rabbit anti-mouse antibody; ab110413; Abcam), vinculin (mouse anti-mouse antibody; V9131; Sigma-Aldrich), β3 integrin (rabbit anti-mouse antibody; #4702; Cell Signaling), or β-actin (rabbit anti-mouse antibody; #4970; Cell Signaling).

Techniques: Control, Activation Assay, Incubation, Magnetic Beads, SDS Page, Mass Spectrometry, Binding Assay, Western Blot, Immunoprecipitation, Inhibition, Expressing, Membrane

Journal: International immunopharmacology

Article Title: Inhibiting the SARM1-NAD + axis reduces oxidative stress-induced damage to retinal and nerve cells.

doi: 10.1016/j.intimp.2024.112193

Figure Lengend Snippet: Fig. 6. DSRM ameliorated oxidative stress-induced neurite injury in N2a cells. (A-C) GOx inhibited N2a cell proliferation and neurite growth in the RA-induced N2a cell differentiation model (magnification 400 × ); (D-E) Immunocytochemistry demonstrated that 10 μM DSRM pre-treatment for 2 h enhanced β3-tubulin expression induced by 7 mU/mL GOx for 12 h in N2a cells (scale bar: 50 μm). N ≥3, **p < 0.01, ****p < 0.0001.

Article Snippet: After culturing and treating the cells in 24-well plates and fixing and permeabilizing them with methanol at -20 ◦C for 15 min, the cells were then washed 3 times with PBS, and then incubated in containment buffer (5 % donkey serum for containment) for 20 min, and then incubated with anti-GSDMD primary antibody (1:500, Abclonal, Wuhan, China, Cat#A20197), anti-β3-tubulin primary antibody (1:1000, CST, USA, Cat#5568S) was incubated at 4 ◦C overnight.

Techniques: Cell Differentiation, Immunocytochemistry, Expressing