Journal: Investigative Ophthalmology & Visual Science

Article Title: Normalization of Wound Healing and Diabetic Markers in Organ Cultured Human Diabetic Corneas by Adenoviral Delivery of c-Met Gene

doi: 10.1167/iovs.09-4569

Figure Lengend Snippet: Antibodies

Article Snippet: Positive In Source/Reference c-Met total Rabbit pAb sc-161 IHC, WB Santa Cruz Biotechnology c-Met total Rabbit pAb sc-10 IHC, WB Santa Cruz Biotechnology c-Met total Rabbit pAb sc-8307 WB * Santa Cruz Biotechnology c-Met total Rabbit pAb 71-8000 Zymed c-Met extracellular Mouse mAb MAB358 WB R&D Systems c-Met extracellular Mouse mAb 05-238 IHC Millipore p-c-Met (Tyr1003) Rabbit pAb ab61024 IHC Abcam p-c-Met (Tyr1234/Tyr1235) Rabbit pAb 44887G WB * Invitrogen p-c-Met (Tyr1230/Tyr1234/Tyr1235) Rabbit pAb 44888G Invitrogen HGF Rabbit pAb sc-7949 IHC Santa Cruz Biotechnology Nidogen-1 Mouse mAb A9 IHC Kabosova et al. 37 Nidogen-1 Mouse mAb MAB2570 IHC R&D Systems Nidogen-2 Rabbit pAb 1080 IHC Kabosova et al. 37 Laminin γ1 chain Rat mAb A5 IHC Kabosova et al. 37 Integrin α 3 β 1 Mouse mAb MAB1992 IHC Millipore Akt Mouse mAb 610860 WB BD Transduction Labs p-Akt (Ser472/Ser473) Mouse mAb 550747 BD Pharmingen p-Akt (Ser473) Rabbit pAb sc-7985-R IHC, WB Santa Cruz Biotechnology p-Akt (Ser473) Rabbit pAb 9271 IHC, WB Cell Signaling p-Akt (Thr308) Rabbit mAb 2965 Cell Signaling ERK1/2 Rabbit mAb 4695 IHC, WB Cell Signaling p-ERK1/2 (Thr202/Tyr204) Rabbit pAb sc-101760 Santa Cruz Biotechnology p-ERK1/2 (Tyr 204) Mouse mAb sc-7383 Santa Cruz Biotechnology p-ERK1/2 (Thr202/Tyr204) Rabbit mAb 4370 WB Cell Signaling p-ERK1/2 (Thr185/Thr202) Rabbit pAb ab4819 IHC Abcam p-ERK1/2 (Thr183/Tyr185) Mouse mAb ab50011 Abcam p38 MAPK Rabbit pAb 9212 WB Cell Signaling p-p38 (Thr180/Tyr182) Rabbit pAb AB3828 WB * Millipore p-p38 (Thr180/Tyr182) Rabbit mAb 9215 WB Cell Signaling p-p38 (Thr180/Tyr182) Mouse mAb 612280 BD Transduction Labs p-p38 (Thr180/Tyr182) Mouse mAb ab50012 IHC Abcam p85α PI3 kinase Mouse mAb sc-56939 Santa Cruz Biotechnology p-p85α PI3 kinase (Tyr508) Goat pAb sc-12929 Santa Cruz Biotechnology p-EGFR (Tyr845) Rabbit pAb 44-784G IHC Invitrogen p-EGFR (Tyr845) Rabbit mAb 2342-1 Epitomics ZO-1 Rabbit pAb 40-2300 IHC † Invitrogen Claudin-1 Rabbit pAb 51-9000 IHC † Invitrogen Ki-67 Mouse mAb sc-101861 IHC ‡ Santa Cruz Biotechnology Activated caspase-3 Rabbit pAb G7481 IHC Promega β-Actin Mouse mAb A5316 WB Sigma-Aldrich Open in a separate window Santa Cruz Biotechnology, Santa Cruz, CA; Zymed, South San Francisco, CA; R&D Systems, Minneapolis, MN; Millipore, Billerica, MA; Abcam, Cambridge, MA; Invitrogen, Carlsbad, CA; BD Transduction Laboratories, Lexington, KY; BD Pharmingen, San Diego, CA; Cell Signaling, Beverly, MA; Epitomics, Burlingame, CA; Promega, Madison, WI; Sigma-Aldrich, St. Louis, MO. mAb, monoclonal antibody; pAb, polyclonal antibody; IHC, immunohistochemistry; WB, Western blot; p-, phosphorylated.

Techniques: Western Blot, Transduction, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: Nidogen 2 Overexpression Promotes Hepatosteatosis and Atherosclerosis

doi: 10.3390/ijms252312782

Figure Lengend Snippet: Expression of NID2 protein is elevated in human atherosclerotic arteries and murine steatotic livers. ( A ) Representative western blot images for NID2 and β-tubulin protein expression in human atherosclerotic inner curvature (IC) and non-atherosclerotic descending aorta (DA) vascular tissue. The bar diagram shows mean protein levels expressed as a ratio of NID2 to β-tubulin. ( B ) Representative Western blot images for NID2 (red arrowhead points to the correct band) and GAPDH in the livers of control diet (CD)- and calorie-matched high-fat diet (HFD, 12 weeks)-fed C57BL/6J mice. The bar diagram represents the mean NID2 protein expression ( n = 4). Statistical analyses were performed using a two-tailed unpaired t -test ( A , B ). Data represent mean ± SEM. * p < 0.05, and **** p < 0.0001.

Article Snippet: The following primary antibodies were used: NID2 (Proteintech, Rosemont, IL, USA #13530-1-AP), total ACC (Cell Signaling Technology, Danvers, MA, USA, #3662S), pACC Ser79 (Cell Signaling Technology, Danvers, MA, USA, #11818S), total AMPKα (Cell Signaling Technology, Danvers, MA, USA, #2793S), pAMPKα Thr172 (Cell Signaling Technology, Danvers, MA, USA, #2535S), IL-6 (Cell Signaling Technology, Danvers, MA, USA, #12912S), TNFα (Cell Signaling Technology, Danvers, MA, USA, #11948T), β-Tubulin (Cell Signaling Technology, Danvers, MA, USA, #86298S), and GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA #sc-365062).

Techniques: Expressing, Western Blot, Control, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Nidogen 2 Overexpression Promotes Hepatosteatosis and Atherosclerosis

doi: 10.3390/ijms252312782

Figure Lengend Snippet: NID2 overexpression enhances liver and epididymal white adipose tissue mass in male mice. ( A ) The schematic diagram illustrates the experimental plan. Apoe −/− mice were injected with control (Ctrl) and NID2 -AAV intraperitoneally, fed a Western diet for 12 weeks, and analyzed. ( B – E ) Male control and NID2 -AAV-injected Apoe −/− mice were utilized to measure NID2 mRNA levels in various organs by qRT-PCR at least in duplicate. Bar diagrams represent mRNA expression in the liver ( B , n = 10), kidney ( C , n = 6), epididymal white adipose tissue (EpiWAT, D , n = 7–10), and heart ( E , n = 10). Bar diagrams show body weight gain ( F ), plasma total cholesterol ( G ), fasting blood glucose ( H ), whole-body fat/lean mass ( I ), liver weight ( J ), adipose tissue weight ( K ), and spleen weight ( L ) ( n = 5–6). A two-tailed unpaired t -test ( C , G – K ), two-tailed unpaired Mann–Whitney test ( B , D , E , L ), and two-way ANOVA followed by Sidak post hoc test for multiple comparisons ( F ) were utilized for statistical analyses. Data represent mean ± SEM. ns: non-significant. * p < 0.05, *** p < 0.001 and **** p < 0.0001.

Article Snippet: The following primary antibodies were used: NID2 (Proteintech, Rosemont, IL, USA #13530-1-AP), total ACC (Cell Signaling Technology, Danvers, MA, USA, #3662S), pACC Ser79 (Cell Signaling Technology, Danvers, MA, USA, #11818S), total AMPKα (Cell Signaling Technology, Danvers, MA, USA, #2793S), pAMPKα Thr172 (Cell Signaling Technology, Danvers, MA, USA, #2535S), IL-6 (Cell Signaling Technology, Danvers, MA, USA, #12912S), TNFα (Cell Signaling Technology, Danvers, MA, USA, #11948T), β-Tubulin (Cell Signaling Technology, Danvers, MA, USA, #86298S), and GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA #sc-365062).

Techniques: Over Expression, Injection, Control, Western Blot, Quantitative RT-PCR, Expressing, Clinical Proteomics, Two Tailed Test, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Nidogen 2 Overexpression Promotes Hepatosteatosis and Atherosclerosis

doi: 10.3390/ijms252312782

Figure Lengend Snippet: NID2 overexpression in mice promotes hepatic lipid accumulation and fibrosis. Male Apoe −/− mice were injected with control and NID2 -AAV intraperitoneally, fed a Western diet for 12 weeks, and analyzed. ( A ) Representative Western blot images for NID2 and GAPDH protein expression in the livers of control and NID2 -overexpressing mice ( n = 3). ( B ) Representative images of liver sections stained with H & E (lipid droplets), ORO (neutral lipid accumulation), and Sirius red (fibrosis); scale bar 100 μm. ( C – H ) Bar diagrams represent lipid accumulation ( C , n = 6), fibrosis area ( D , n = 5), hepatic triglyceride ( E , n = 3–4), NEFA levels ( F , n = 3–4), plasma triglyceride ( G , n = 5) and NEFA levels ( H , n = 5), in control and NID2 -AAV-injected mice. Statistical analyses were performed using a two-tailed unpaired t -test ( C – H ). Data represent mean ± SEM. ns: non-significant. * p < 0.05, and ** p < 0.01.

Article Snippet: The following primary antibodies were used: NID2 (Proteintech, Rosemont, IL, USA #13530-1-AP), total ACC (Cell Signaling Technology, Danvers, MA, USA, #3662S), pACC Ser79 (Cell Signaling Technology, Danvers, MA, USA, #11818S), total AMPKα (Cell Signaling Technology, Danvers, MA, USA, #2793S), pAMPKα Thr172 (Cell Signaling Technology, Danvers, MA, USA, #2535S), IL-6 (Cell Signaling Technology, Danvers, MA, USA, #12912S), TNFα (Cell Signaling Technology, Danvers, MA, USA, #11948T), β-Tubulin (Cell Signaling Technology, Danvers, MA, USA, #86298S), and GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA #sc-365062).

Techniques: Over Expression, Injection, Control, Western Blot, Expressing, Staining, Clinical Proteomics, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Nidogen 2 Overexpression Promotes Hepatosteatosis and Atherosclerosis

doi: 10.3390/ijms252312782

Figure Lengend Snippet: NID2 overexpression augments atherosclerosis in male hypercholesterolemic mice. Male Apoe −/− mice were injected with control (Ctrl) and NID2 -AAV intraperitoneally, fed a Western diet for 12 weeks and analyzed. ( A ) Representative in situ images of the aortic arch (red arrowheads point to atherosclerotic lesions). ( B ) Representative ORO staining of whole aortas; scale bar 5 mm. The bar diagram represents ORO-positive areas in whole aortas ( n = 6). ( C ) Representative images of aortic root cross-sections stained with H & E (lesion area and necrotic core), ORO (lipid accumulation), and Masson’s trichrome (collagen content); scale bar 200 μm. ( D – G ) Bar diagrams show lesion area ( D ), lipid deposition ( E ), collagen content ( F ), and necrotic core area ( G ) ( n = 5–6). Statistical analyses were performed using a two-tailed unpaired t -test ( B , D – G ). Data represent mean ± SEM. * p < 0.05, and ** p < 0.01.

Article Snippet: The following primary antibodies were used: NID2 (Proteintech, Rosemont, IL, USA #13530-1-AP), total ACC (Cell Signaling Technology, Danvers, MA, USA, #3662S), pACC Ser79 (Cell Signaling Technology, Danvers, MA, USA, #11818S), total AMPKα (Cell Signaling Technology, Danvers, MA, USA, #2793S), pAMPKα Thr172 (Cell Signaling Technology, Danvers, MA, USA, #2535S), IL-6 (Cell Signaling Technology, Danvers, MA, USA, #12912S), TNFα (Cell Signaling Technology, Danvers, MA, USA, #11948T), β-Tubulin (Cell Signaling Technology, Danvers, MA, USA, #86298S), and GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA #sc-365062).

Techniques: Over Expression, Injection, Control, Western Blot, In Situ, Staining, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Nidogen 2 Overexpression Promotes Hepatosteatosis and Atherosclerosis

doi: 10.3390/ijms252312782

Figure Lengend Snippet: NID2 overexpression inhibits the activation of the lipid metabolism-related protein AMPK. ( A ) Representative Western blot images for lipid metabolism and pro-inflammatory proteins utilizing liver lysates from control and NID2 -AAV-injected mice. Bar diagrams represent mean protein expression ( B , C ) as the ratios of phospho-total proteins ACC ( B ) and AMPK ( C ), and protein levels of IL-6 ( D ) and TNFα ( E ) ( n = 5). Statistical analyses were performed using a two-tailed unpaired t -test. Data represent mean ± SEM. ns: non-significant. * p < 0.05.

Article Snippet: The following primary antibodies were used: NID2 (Proteintech, Rosemont, IL, USA #13530-1-AP), total ACC (Cell Signaling Technology, Danvers, MA, USA, #3662S), pACC Ser79 (Cell Signaling Technology, Danvers, MA, USA, #11818S), total AMPKα (Cell Signaling Technology, Danvers, MA, USA, #2793S), pAMPKα Thr172 (Cell Signaling Technology, Danvers, MA, USA, #2535S), IL-6 (Cell Signaling Technology, Danvers, MA, USA, #12912S), TNFα (Cell Signaling Technology, Danvers, MA, USA, #11948T), β-Tubulin (Cell Signaling Technology, Danvers, MA, USA, #86298S), and GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA #sc-365062).

Techniques: Over Expression, Activation Assay, Western Blot, Control, Injection, Expressing, Two Tailed Test

Journal: Investigative Ophthalmology & Visual Science

Article Title: Descemet's Membrane Modulation of Posterior Corneal Fibrosis

doi: 10.1167/iovs.18-26451

Figure Lengend Snippet: Nidogen-1 and SMA localization 4 weeks after posterior corneal injury. Duplex IHC for nidogen-1 and SMA was performed in all panels. (A) In an unwounded control cornea, nidogen-1 (green) is present at high levels in the epithelial basement membrane (arrows), Descemet's basement membrane (arrowheads), and at relatively higher levels in the posterior stroma than anterior stroma, as was previously reported. Epithelial cells and corneal endothelial cells also express nidogen-1. Note there were no SMA + cells in the unwounded corneas. e, epithelium in all panels where it is present. Blue is DAPI staining of nuclei in all panels. Magnification, 100×. (B) At 4 weeks after excision of the Descemet's membrane–endothelial complex over the central 8 mm of the cornea, there was a zone of fibrosis (f) populated with SMA + (red) myofibroblasts that occupied the posterior approximately 30% to 40% of the corneal stroma in all six corneas examined. In this panel, the periphery of the initial injury and resulting fibrosis is seen. Note there was low levels of nidogen-1 (green, at arrows) within the fibrous zone. Superior to the fibrous zone, and penetrating into it on the left, were SMA − cells that had high levels of nidogen-1. There appeared to be peripheral fragments of basement membrane (arrowheads) above the fibrosis that was only noted at the edge of the wound where possibly the fibrosis had extended peripheral to the Descemet's membrane injury. The cells in the stroma above the fibrosis could be keratocytes, corneal fibroblasts, fibrocytes, or other infiltrating corneal endothelial cells. Magnification, 400×. (C) In the central cornea at 4 weeks after excision of the Descemet's membrane–endothelial complex, a zone of fibrosis (f) populated with SMA + (red) myofibroblasts occupied the posterior stroma in all six corneas in this group. Again, low levels of nidogen-1 (arrows) were detected within this fibrosis. Overlying the fibrosis, cells that likely include corneal fibroblasts and fibrocytes (arrowheads, also see B) with high levels of nidogen-1. Wavy linear deposits of nidogen-1 are also seen in this area of the stroma above the fibrosis. Magnification, 400×. (D) A no nidogen-1 or SMA primary antibody control IHC staining of a cornea that had excision of the Descemet's membrane–endothelial complex at 1 month after surgery. Magnification, 100×. (E) IHC for nidogen-1 and SMA at 4 weeks after removal of the endothelium alone over the central 8 mm of the cornea. There were no SMA + cells, and the normal distribution of nidogen-1 was present (compare with A) after regeneration of the corneal endothelium (arrowheads) in all corneas in this group, although nidogen-1 was possibly at lower levels in some areas of the stroma than in the unwounded control. Magnification, 100×.

Article Snippet: SMA and nidogen-1 double-IHC was performed using the same method except the primary antibody for SMA was goat anti-SMA (Cat. # NB300-978; Novus Biologicals USA, Littleton, CO, USA) used at 1:100 dilution for 60 minutes, and the primary antibody for nidogen-1 was goat anti-human nidogen-1/entactin antigen affinity-purified polyclonal antibody (Catalog #AF 2570; R&D Systems, Minneapolis, MN, USA) used at 1:50 dilution and incubated for 90 minutes.

Techniques: Membrane, Staining, Immunohistochemistry