Journal: Investigative Ophthalmology & Visual Science

Article Title: Myocilin Regulates Metalloprotease 2 Activity Through Interaction With TIMP3

doi: 10.1167/iovs.16-20336

Figure Lengend Snippet: TIMP3 is identified as a myocilin-binding protein. (A) Western blot analysis of immunoprecipitates with anti-myocilin antibodies or rabbit IgG from eye lysates of Myoc null (KO), WT, and Tg mice expressing WT (wt MYOC) or mutated mouse myocilin (m MYOC). The efficiency and specificity of immunoprecipitation were confirmed using anti-myocilin antibodies. The peptide number (N) indicates the number of peptides associated with myocilin (MYOC) or TIMP3 identified in the immunoprecipitates by shotgun proteomics. (B) Myocilin interacts with TIMP3 in HEK293 cells. HEK293 cells were cotransfected with indicated HA-tagged human TIMP constructs and a FLAG-tagged human myocilin construct. The lysates were immunoprecipitated with anti-HA antibodies and the precipitates were probed with anti-FLAG antibodies. (C) Myocilin interacts with TIMP3 in HepG2 cells. HepG2 cells were cotransfected with a myc-tagged TIMP3 construct and indicated FLAG-tagged myocilin constructs. The lysates were immunoprecipitated with anti-FLAG antibodies and the precipitates were probed with anti-myc antibodies. The arrowhead marks TIMP3 band and the arrow marks immunoglobulin band. (D) Model of TIMP3–OLF interaction based on computational docking, suggesting that OLF does not compete for the MMP binding site on TIMP3, which is near its N-terminus.

Article Snippet: Sections were fixed with 4% PFA for 10 minutes on ice, blocked with Blocker Casein PBS (Thermo Fisher) for 30 minutes at room temperature, and then incubated with primary antibodies against mouse myocilin or TIMP3 (Novus Biologicals) at 4°C overnight.

Techniques: Binding Assay, Western Blot, Expressing, Immunoprecipitation, Construct

Journal: Investigative Ophthalmology & Visual Science

Article Title: Myocilin Regulates Metalloprotease 2 Activity Through Interaction With TIMP3

doi: 10.1167/iovs.16-20336

Figure Lengend Snippet: Distribution of myocilin and TIMP3 in the ocular tissues. Cryosections of the FVRD mouse eyes were stained with anti-myocilin and TIMP3 antibodies. (A–C, G–I) Eye drainage structures: (A) Myocilin is detected mainly in the TM. (B) The image of the same area as in (A) obtained using DIC microscopy for better visualization of the tissue orientation. (C) Enlarged image of the TM area boxed in (A). (G) TIMP3 is detected in the TM and ciliary body. (H) The image of the same area as in (D) obtained using DIC microscopy. (H) Enlarged image of the TM area boxed in (G). (D–F, J–L) Posterior part of the eye: (D) Myocilin was detected in the choroid and sclera. (E) The image of the same area as in (D) obtained using DIC microscopy. (F) Enlarged image of the RPE–sclera region. (J) TIMP3 was detected in the RPE, Bruch's membrane, choroid, and sclera. (K) The image of the same area as in (J) obtained using DIC microscopy. (L) An enlarged image of RPE–sclera region. Abbreviations are as in . BrM, Bruch's membrane.

Article Snippet: Sections were fixed with 4% PFA for 10 minutes on ice, blocked with Blocker Casein PBS (Thermo Fisher) for 30 minutes at room temperature, and then incubated with primary antibodies against mouse myocilin or TIMP3 (Novus Biologicals) at 4°C overnight.

Techniques: Staining, Microscopy, Membrane

Journal: Investigative Ophthalmology & Visual Science

Article Title: Myocilin Regulates Metalloprotease 2 Activity Through Interaction With TIMP3

doi: 10.1167/iovs.16-20336

Figure Lengend Snippet: Myocilin enhances the inhibitory activity of TIMP3 toward MMP2. (A) The purity of TIMP3 and myocilin was estimated by SDS-PAGE. (B) MMP2 (50 nM) was coincubated with indicated different concentrations of TIMP3. The fluorescence signals representing the protease activity of MMP2 were monitored for 2 hours. Arrow indicates TIMP3 concentration selected for further studies. (C) Myocilin (200 nM) or control IgG (200 nM) was preincubated with TIMP3 (100 nM). TIMP3 alone or the protein mixtures were added to the MMP2 proteolytic reaction. Error bars represent ±SD of triplicate reactions. Statistically significant differences between two groups (+ MMP2, TIMP3 and + MMP2, TIMP3, MYOC) at different time points are indicated by asterisks (P < 0.05).

Article Snippet: Sections were fixed with 4% PFA for 10 minutes on ice, blocked with Blocker Casein PBS (Thermo Fisher) for 30 minutes at room temperature, and then incubated with primary antibodies against mouse myocilin or TIMP3 (Novus Biologicals) at 4°C overnight.

Techniques: Activity Assay, SDS Page, Fluorescence, Concentration Assay, Control

Journal: British Journal of Pharmacology

Article Title: β 3 ‐Adrenoceptor as a potential immuno‐suppressor agent in melanoma

doi: 10.1111/bph.14660

Figure Lengend Snippet: (a) Representative images of mouse spleens at T7 (n = 6; left) and mean weight of mouse spleens (right). (b) FACS analysis and quantification at T7 and T14 of NK (NKp46+/NK1.1+ gated on CD3−/CD45+). (c) FACS analysis and quantification at T7 and T14 of CD8+ (gated on CD45+). (d) FACS analysis and quantification at T7 and T14 of perforin expression on NKp46+/NK1.1+ cells. Two‐way ANOVA analysis was performed. (e) FACS analysis and quantification at T7 and T14 of CD8+ cytotoxic (CD107+ gated on CD8+). (f) FACS analysis and quantification at T14 of NK (NKp46+/NK1.1+ gated on CD3−/CD45+) cells in siRNA‐CTRL, siRNA‐β2, and siRNA‐β3 treated mice (n = 6). (g) FACS analysis and quantification at T14 of CD8+ (gated on CD45+) cells in siRNA‐CTRL, siRNA‐β2, and siRNA‐β3 treated mice (n = 6). *P < 0.05 Prop‐ (or siRNA‐β2) compared with Veh‐; # P < 0.05 SR‐ (or siRNA‐β3) compared with Veh‐; $ P < 0.05 SR‐ (or siRNA‐β3) compared with Prop‐ (or siRNA‐β2)

Article Snippet: Cells isolated from mouse tumours, spleens, and blood were incubated and stained with appropriate dilutions of various combinations of the following fluorochrome‐conjugated antibodies: anti‐CD 45‐VioBlue (Miltenyi Biotec Cat# 130‐092‐880, RRID:AB_1103220) or VioGreen (Miltenyi Biotec Cat# 130‐096‐906, RRID:AB_2660419), anti‐NKp46‐FITC (Miltenyi Biotec Cat# 130‐102‐300, RRID:AB_2661345), anti‐CD8a‐APC Vio 770 (Miltenyi Biotec Cat# 130‐102‐305, RRID:AB_2659897), anti‐CD3e (17A 2 )‐PE Vio 770 (Miltenyi Biotec Cat# 130‐105‐461, RRID:AB_2657921), anti‐CD107‐PE (Miltenyi Biotec Cat# 130‐111‐318, RRID:AB_2654464), anti‐CD161(NK1.1)‐PerCP Vio700 (Miltenyi Biotec Cat# 130‐117‐773, RRID:AB_2728038), anti‐CD25‐PE (Miltenyi Biotec Cat# 130‐102‐593, RRID:AB_2660259), anti‐CD4‐FITC (Miltenyi Biotec Cat# 130‐102‐541, RRID:AB_2659902), anti‐CD127‐APC (Miltenyi Biotec Cat# 130‐110‐274, RRID:AB_2654842), anti‐CD11b‐PerCP Vio700 (Miltenyi Biotec Cat# 130‐109‐289, RRID:AB_2654659), anti‐Gr1‐PE (Miltenyi Biotec Cat# 130‐102‐426, RRID:AB_2659861), anti‐ CD95 (Fas) ‐APC (Miltenyi Biotec Cat# 130‐106‐907, RRID:AB_2659651), anti‐F4/80‐PerCP Vio700 (Miltenyi Biotec Cat# 130‐102‐161, RRID:AB_2651711), anti‐CD16/32‐VioBright FITC (Miltenyi Biotec Cat# 130‐108‐364, RRID:AB_2660221), anti‐ CD11c (integrin, α X subunit)‐APC Vio770 (Miltenyi Biotec Cat# 130‐107‐461, RRID:AB_2660162), anti‐ IL‐10 ‐APC (Miltenyi Biotec Cat# 130‐102‐349, RRID:AB_2660626), anti‐ integrin α 7 ‐APC (Miltenyi Biotec Cat# 130‐102‐717, RRID:AB_2652466), anti‐ iNOS ‐APC (Santa Cruz Biotechnology Cat# sc‐7271, RRID:AB_627810), anti‐ Arg1 ‐FITC (R and D Systems Cat# IC5868F, RRID:AB_10718118), anti‐β 2 ‐FITC (Biorbyt Cat# orb15065, RRID:AB_10735676), anti‐β 3 ‐PE (Biorbyt Cat# orb124479, RRID:AB_2783863), or PerCP Vio700 (Biorbyt Cat# orb123003, RRID:AB_2783864).

Techniques: Expressing