vitronectin  (R&D Systems)

Journal: bioRxiv

Article Title: Loss of Lamp2a-dependent chaperone-mediated autophagy drives dry AMD-like retinal pathology in mice and is rescued by BK channel activation

doi: 10.64898/2026.03.19.712761

Figure Lengend Snippet: (A) Representative confocal immunofluorescence images showing ApoE and PLIN2 staining (green). Scale bar = 30 µm. Lamp2a -/- mice showed increased staining of both proteins compared with WT mice, which was markedly reduced by GLA-1-1 treatment. (B) Bar graph showing quantification of ApoE fluorescence intensity (mean gray value) (n = 7 mice). (C) Bar graph showing quantification of PLIN2 immunofluorescence signal (n = 5 mice). (D) Representative confocal immunofluorescence images showing staining for vitronectin, MMP2, and clusterin. Scale bar = 30 µm. Compared with WT mice, Lamp2a -/- mice showed markedly increased immunoreactivity for these proteins, which was significantly reduced by GLA-1-1 treatment. (E) Bar graph showing quantification of MMP2 fluorescence intensity (mean gray value) (n=5 mice). (F) Bar graph showing quantification of vitronectin fluorescence intensity (mean gray value) (n=7 mice). (G) Bar graph showing quantification of clusterin fluorescence intensity (mean gray value) (n=7 mice). (H) Representative immunoblots showing protein levels of ApoE, clusterin, vitronectin, and PLIN2 in RPE/choroid lysates. Bar graphs showing immunoblot quantification of protein levels of vitronectin (I), clusterin (J), ApoE (K), and PLIN2 (L) in RPE/choroid lysates (n = 3 mice per group). Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Values are expressed as mean ± SD.

Article Snippet: Antibodies against LC3B (Sigma, L8918-200UL), actin (Proteintech, 66009-1-Ig), PLIN2 (Proteintech, 15294-1-AP), fibronectin (Invitrogen, PA5-29578), clusterin (R&D systems, AF2747), vitronectin (R&D Systems, MAB38751), and p62 (Abnova, H00008878-M01; CST, 5114), Lamp2a (Abcam, ab125068), Lamp2b (Abcam, ab13524), Iba1 (Fujifilm Wako, 019-19741), Laminin (Sigma, L9393) and ApoE (Abcam, ab183596) were used in this study.

Techniques: Immunofluorescence, Staining, Fluorescence, Western Blot

Journal: bioRxiv

Article Title: Vitronectin defines a differentiation-restraining extracellular matrix state in myogenic cells

doi: 10.64898/2026.01.14.699461

Figure Lengend Snippet: a, Immunoblot analysis of VN protein levels in fetal bovine serum, adult bovine serum, and adult horse serum. b, Immunoblot analysis of VN protein on day 0 (d0; before differentiation induction), day 1, and day 3 (d1 and d3; 1 and 3 day after differentiation induction). c, Representative immunofluorescence images showing the level and localization of VN protein in growth medium (GM) and day 1 in differentiation medium (DM). d, qPCR analysis for transcription of VN gene on d0, d1, and d3 after differentiation induction (n = 3). e, Immunoblot analysis for confirmation of immunodepleting of vitronectin from FBS. Vitronectin was depleted from FBS using control and anti-vitronectin antibody immobilized on Protein A Sepharose. f, Quantification of the inhibitory effect on myoblast differentiation with control and VN-immunodepleted FBS by immunofluorescence for MHC. (Control FBS: n = 1688, VN-reduced FBS: n = 1663 in counted nuclei number). g, Representative phase-contrast images of C2C12 cells cultured in GM, DM, and a serum-free DA-X medium supplemented with LIF on plates coated with or without VN coating. Images were captured on day 5 in each condition. Scale bar: 100 µm. Data are presented as mean ± s.d. Statistical significance was determined using one-way ANOVA in d and Student’s t-test in f ; * p < 0.05 .

Article Snippet: As a first antibody, Anti-MyoD (5.8A, 1:5000; Novus Biologicals, NB100-56511), anti-Myogenin (1:10000; Abcam, ab124800), Anti-MHC (MF20, 1:5000; R&D Systems, MAB4470), Anti-β-Actin (C4, 1:5000; Santa Cruz, sc-47778), Anti-Stat3 (D3Z2G, 1:5000; Cell Signaling Technology, #12640), Anti-phospho-Stat3 (Tyr705, D3A7, 1:5000; Cell Signaling Technology, #9145), Anti-ERK1/2 (C-9, 1:5000; Santa Cruz, sc-514302), Anti-phospho-ERK (E-4, 1:5000; Santa Cruz, sc-7383), Anti-p38α/β MAPK (A-12, 1:5000; Santa Cruz, sc-7972), anti-phospho-p38 (E-1, 1:5000; Santa Cruz, sc-166182), Anti-p21 Waf/Cip1 (F-5, 1:5000; Santa Cruz, sc-6246), Anti-p27 Kip2 (F-8, 1:5000; Santa Cruz, sc-1641), Anti-FGF Receptor 1 (D8E4, 1:5000; Cell Signaling Technology, #9740), Anti-p-FGF Receptor 1 (Try653/654) (D4X3D, 1:5000; Cell Signaling Technology, #52928), Anti-Insulin Receptor β (4B8, 1:5000; Cell Signaling Technology, #3025), Anti-p-IGF-1 Receptor β (Tyr1135/1136)/Insulin Receptor β (Try1150/1151) (19H7, 1:5000; Cell Signaling Technology, #3024), Anti-Vitronectin 65/75 (D-8, 1:5000; Santa Cruz, sc-74484), Anti-p16 INK4A (F-12, 1:5000; Santa Cruz, sc-1661), p-χHistone H2A.X (Ser 139, 1:5000; Santa Cruz, sc-517348).

Techniques: Western Blot, Immunofluorescence, Control, Cell Culture

Journal: bioRxiv

Article Title: Vitronectin defines a differentiation-restraining extracellular matrix state in myogenic cells

doi: 10.64898/2026.01.14.699461

Figure Lengend Snippet: a, Schematic representation of the myogenic differentiation processes in C2C12 myoblasts dependent on culture medium and expression dynamics of representative muscle differentiation markers. Proliferation of myoblasts requires growth medium (GM; 10% fetal bovine serum (FBS) supplemented medium), whereas differentiation medium (DM; 2% horse serum (HS) supplemented medium) induces rapid differentiation into multinucleated myotubes and loss of proliferative capacity. b, Representative phase contrast and immunofluorescence images showing expression of the differentiation marker Myogenin on day 3 in DM. White arrowheads indicate representative differentiating myotubes. Differentiation index quantified by the proportion of myogenin-positive cells relative to total nuclei is shown in the graph. (Control: n = 1652, Vitronectin: n = 1746) c, Time-course observation of myotube differentiation by immunofluorescence for myosin heavy chain (MHC). d, Fusion index quantified by the number of nuclei per MHC-positive myotube on day 3 in DM shown in the graph (Control: n=50, Vitronectin: n=50 in MHC-positive cells). The numbers shown in the immunofluorescence images indicate of nuclei in the representative MHC-positive myotube. e, Immunoblot analysis of C2C12 cells for the representative myogenic differentiation markers: MyoD, Myogenin, and MHC. f, Immunoblot analysis of C2C12 cells for non- and phosphorylated form of STAT3, ERK1/2, and p38 MAPK. g-h , Immunofluorescence analysis of MHC expression after 3 days in DM under the indicated conditions: control, VN, VN plus cRGDfV, and cRGDfV alone. Quantification shows the proportion of MHC-positive cells relative to total nucle as differentiation indexi (g) and fusion index (h). Scale bar: 100 µm. Nuclei were counterstained with DAPI. Data in the graphs are presented as mean ± s.d. Statistical significance determined using Student’s t-test; ** p < 0.01 .

Article Snippet: Beads were then incubated with 10 μL of anti-vitronectin 65/75 antibody (D-8; Santa Cruz, sc-74484) and control antibody (Santa Cruz, sc-3877) overnight at 4°C.

Techniques: Cell Characterization, Expressing, Immunofluorescence, Marker, Control, Western Blot

Journal: bioRxiv

Article Title: Vitronectin defines a differentiation-restraining extracellular matrix state in myogenic cells

doi: 10.64898/2026.01.14.699461

Figure Lengend Snippet: a, Schematic representation of the experimental workflow to evaluate the proliferative capacity of C2C12 myoblasts cultured at low cell density in supplementation with vitronectin (GM: growth medium, DM: differentiation medium). b, Representative nuclei images stained with DAPI and the relative cell number of C2C12 cells counted on day 3 of differentiation shown in the graph (n = 6). c, Representative immunofluorescence images for phospho-Histone H3 (pH3) on day 3 of differentiation and the proportion of pH3-positive cells relative to total nuclei shown in the graph (Control: n = 1345, Vitronectin: n = 2632). d, Representative immunofluorescence images for cleaved caspase-3 on day 2 of differentiation. The ratio of apoptotic cells relative to total nuclei is shown in the graph (Control: n = 2499, Vitronectin: n = 2345). e, Western blot analysis of C2C12 cells for p21 and p27, cell cycle inhibitors. f, Immunoblot analysis of C2C12 cells for non- and phosphorylated insulin-like growth factor receptor (IGFR) and fibroblast growth factor receptor (FGFR), key growth factor receptors involved in the proliferation of C2C12 cells. Scale bar: 100 µm. White arrowheads indicate representative positive cells for p-Histone H3 (c) and cleaved caspace-3 (d). Data are presented as mean ± s.d. Statistical significance determined using Student’s t-test; ** p < 0.01 , * p < 0.05 .

Article Snippet: Beads were then incubated with 10 μL of anti-vitronectin 65/75 antibody (D-8; Santa Cruz, sc-74484) and control antibody (Santa Cruz, sc-3877) overnight at 4°C.

Techniques: Cell Culture, Staining, Immunofluorescence, Control, Western Blot

Journal: bioRxiv

Article Title: Vitronectin defines a differentiation-restraining extracellular matrix state in myogenic cells

doi: 10.64898/2026.01.14.699461

Figure Lengend Snippet: a, Schematic representation of the rotary suspension spheroid culture system. C2C12 cells were first aggregated under static conditions in a low-attachment U-shaped 96-well plate for 1 day, then transferred to suspension culture on an orbital shaker inside an incubator (37 °C, 150 rpm.) for another 5 days. b, Quantification of total cell numbers per spheroid on day 6 (n = 5). c, Representative phase-contrast and immunofluorescence images of spheroid for MHC and DAPI. Scale bar: 100 µm. d, Representative confocal images of spheroid stained for MHC, Ki-67, and DAPI on day 6. Quantification of the proportion of MHC- or Ki-67-positive cells relative to total nuclei is shown in the graphs (Control: n = 1537, Vitronectin: n = 1522). Scale bar: 50 µm. Data are presented as mean ± s.d. Statistical significance was determined using Student’s t-test; ** p < 0.01 .

Article Snippet: Beads were then incubated with 10 μL of anti-vitronectin 65/75 antibody (D-8; Santa Cruz, sc-74484) and control antibody (Santa Cruz, sc-3877) overnight at 4°C.

Techniques: Suspension, Immunofluorescence, Staining, Control

Journal: bioRxiv

Article Title: Vitronectin defines a differentiation-restraining extracellular matrix state in myogenic cells

doi: 10.64898/2026.01.14.699461

Figure Lengend Snippet: a, Immunoblot analysis of VN protein levels in fetal bovine serum, adult bovine serum, and adult horse serum. b, Immunoblot analysis of VN protein on day 0 (d0; before differentiation induction), day 1, and day 3 (d1 and d3; 1 and 3 day after differentiation induction). c, Representative immunofluorescence images showing the level and localization of VN protein in growth medium (GM) and day 1 in differentiation medium (DM). d, qPCR analysis for transcription of VN gene on d0, d1, and d3 after differentiation induction (n = 3). e, Immunoblot analysis for confirmation of immunodepleting of vitronectin from FBS. Vitronectin was depleted from FBS using control and anti-vitronectin antibody immobilized on Protein A Sepharose. f, Quantification of the inhibitory effect on myoblast differentiation with control and VN-immunodepleted FBS by immunofluorescence for MHC. (Control FBS: n = 1688, VN-reduced FBS: n = 1663 in counted nuclei number). g, Representative phase-contrast images of C2C12 cells cultured in GM, DM, and a serum-free DA-X medium supplemented with LIF on plates coated with or without VN coating. Images were captured on day 5 in each condition. Scale bar: 100 µm. Data are presented as mean ± s.d. Statistical significance was determined using one-way ANOVA in d and Student’s t-test in f ; * p < 0.05 .

Article Snippet: Beads were then incubated with 10 μL of anti-vitronectin 65/75 antibody (D-8; Santa Cruz, sc-74484) and control antibody (Santa Cruz, sc-3877) overnight at 4°C.

Techniques: Western Blot, Immunofluorescence, Control, Cell Culture

Journal: bioRxiv

Article Title: Vitronectin defines a differentiation-restraining extracellular matrix state in myogenic cells

doi: 10.64898/2026.01.14.699461

Figure Lengend Snippet: a, Representative immunofluorescence images for MHC showing myotube maturation. Maturation was quantified by the number of nuclei per MHC-positive myotube (Control: n=100, Vitronectin: n = 100 in MHC-positive cells). Scale bar: 50 µm. b, The relative cell number of chick myogenic cells cultured in indicated conditions (n = 3). c, Representative immunofluorescence images for phosphorylated histone H3 (p-Histone H3) and the proportion of positive cells relative to total nuclei (Control: n = 7619, Vitronectin: n = 7928). Scale bar: 50 µm. d, Representative phase-contrast and nuclei images of spheroid on day 5 in rotary suspension spheroid culture. Scale bar: 100 µm. In a - c , cells were analyzed 6 h after differentiation induction in DM. Nuclei were counterstained with DAPI. Data are presented as mean ± standard deviation. Statistical significance was determined using Student’s t-test; ** p < 0.01 .

Article Snippet: Beads were then incubated with 10 μL of anti-vitronectin 65/75 antibody (D-8; Santa Cruz, sc-74484) and control antibody (Santa Cruz, sc-3877) overnight at 4°C.

Techniques: Immunofluorescence, Control, Cell Culture, Suspension, Standard Deviation