rat anti mouse plvap meca32 monoclonal antibody mab meca 32 hybridoma  (Developmental Studies Hybridoma Bank)

Journal: Clinical Cancer Research

Article Title: PERK Inhibition by HC-5404 Sensitizes Renal Cell Carcinoma Tumor Models to Antiangiogenic Tyrosine Kinase Inhibitors

doi: 10.1158/1078-0432.CCR-23-1182

Figure Lengend Snippet: HC-5404 sensitizes 786-O xenografts to the antiangiogenic effects of axitinib. A, IHC images of 786-O xenograft sections stained with antibodies specific to Meca32, CD31, and SMA. Scale bar = 150 μm. HC-5404 and axitinib administered at 30 mg/kg, twice-a-day for 7 days. B–D, Quantification of IHC staining of xenograft sections. Graphs indicate proportion of cells stained positive for Meca32, positive for CD31 in absence of SMA (%CD31 + SMA − ), or cells that stain positive for both CD31 and SMA (%CD31 + SMA + ). One-way ANOVA statistical analysis evaluated differences between treatment groups; asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; ****, P < 0.001).

Article Snippet: Primary antibodies used were as follows: α-CD31 (Abcam, catalog No. ab182981, RRID:AB_2920881), α-smooth muscle actin (SMA; Abcam, catalog No. ab124964, RRID:AB_11129103), and α-Meca32 (Novus Biologicals, catalog No. NB100–77668, RRID:AB_2276108).

Techniques: Staining, Immunohistochemistry

Journal: Clinical Cancer Research

Article Title: PERK Inhibition by HC-5404 Sensitizes Renal Cell Carcinoma Tumor Models to Antiangiogenic Tyrosine Kinase Inhibitors

doi: 10.1158/1078-0432.CCR-23-1182

Figure Lengend Snippet: Addition of HC-5404 to axitinib regimen induces tumor regression. A, 786-O tumor growth across the study period. Xenografts that progressed in presence of axitinib for 14 days were rerandomized and transferred to indicated treatment groups for an additional 28 days. Data represent mean ± S.E.M. Final tumor volume analyzed by Welch ANOVA. Data represent mean ± S.E.M., n = 8 mice per group (**, P < 0.01; ***, P < 0.005; ****, P < 0.001). B, IHC images of xenograft sections stained with antibodies specific for CD31 and SMA. C, Quantification of proportion of cells that stained positive for Meca32, CD31+SMA- (immature blood vessels), or CD31 + SMA + (mature blood vessels) in B . One-way ANOVA was used to evaluate the statistical significance between groups. Asterisks indicate statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.001). D, Quantification of IHC staining of tumor sections using antibodies specific for the pericyte markers NG2 and MCAM. One-way ANOVA statistical analysis evaluated differences between treatment groups; asterisks indicate significant differences (**, P < 0.01; ***, P < 0.005; ****, P < 0.001).

Article Snippet: Primary antibodies used were as follows: α-CD31 (Abcam, catalog No. ab182981, RRID:AB_2920881), α-smooth muscle actin (SMA; Abcam, catalog No. ab124964, RRID:AB_11129103), and α-Meca32 (Novus Biologicals, catalog No. NB100–77668, RRID:AB_2276108).

Techniques: Staining, Immunohistochemistry

Journal: Cell reports

Article Title: Fibroblasts repair blood-brain barrier damage and hemorrhagic brain injury via TIMP2

doi: 10.1016/j.celrep.2022.111709

Figure Lengend Snippet: (A) Schematic illustration of the in vitro ICH model. (B) TEER values of the in vitro ICH model in the presence or absence of fibroblasts. n = 6 biological replicates. ***p = 0.0002 by Student’s t test. (C) Quantification of 4-kDa FITC-dextran leakage in the in vitro ICH model in the presence or absence of fibroblasts. n = 6 biological replicates. **p = 0.0016 and **p = 0.0060 for 30- and 60-min time points by Student’s t test, respectively. (D) Representative images of ZO-1 (green), claudin5 (green), and DAPI (blue) in primary human brain microvascular endothelial cells (HBMECs) under normal conditions (uninjured) and at 48 h after in vitro ICH with or without fibroblasts. Scale bars, 25 μm. (E) Representative images of caveolin-1 (green), meca32 (red), and DAPI (blue) in primary HBMECs under normal conditions (uninjured) and at 48 h after in vitro ICH with or without fibroblasts. Scale bars, 25 μm. (F) Pie chart showing the percentage of each category of proteins relative to total fibroblast-derived proteins identified by LC-MS/MS. (G) TEER values of the in vitro ICH model in the presence of mouse IgG (control), PAI1 function-blocking antibody, and TIMP2 function-blocking antibody. n = 6 biological replicates. **p = 0.0088 by Student’s t test. (H) Quantification of 4-kDa FITC-dextran leakage in the in vitro ICH model in the presence of mouse IgG (control), PAI1 function-blocking antibody, and TIMP2 function-blocking antibody. n = 6 biological replicates, ***p = 0.00013 and ****p < 0.0001 at 30- and 60-min time points by Student’s t test, respectively. (I) Representative western blot image and quantification of TIMP2 expression secreted by fibroblasts after transduction of lentivirus-expressing TIMP2 short hairpin RNA (shRNA) or a scramble sequence (control). n = 5 biological replicates. *p = 0.0121 by Mann-Whitney U test. (J) TEER values of the in vitro ICH model in the presence of control or TIMP2-knockdown fibroblasts. n = 6 biological replicates. **p = 0.0017 by Student’s t test. (K) Quantification of 4-kDa FITC-dextran leakage in the in vitro ICH model in the presence of control or TIMP2-knockdown fibroblasts. n = 6 biological replicates. ****p < 0.0001 by Student’s t test. (L) Schematic illustration of in vitro TIMP2 rescue experiments. (M) TEER values of the in vitro ICH model in the presence of saline (control) or recombinant TIMP2 protein. n = 6 biological replicates. **p = 0.0028 by Student’s t test. (N) Quantification of 4-kDa FITC-dextran leakage in the in vitro ICH model in the presence of saline (control) or recombinant TIMP2 protein. n = 6 biological replicates. ***p = 0.0006 and ***p = 0.0007 at 30- and 60-min time points by Student’s t test, respectively. (O) Representative images of ZO-1 (green), claudin5 (green), and DAPI (blue) in primary HBMECs treated with saline (control) or recombinant TIMP2 protein at 48 h after in vitro ICH. Scale bars, 25 μm. (P) Representative images of caveolin-1 (green), meca32 (red), and DAPI (blue) in primary HBMECs treated with saline (control) or recombinant TIMP2 protein at 48 h after in vitro ICH. Scale bars, 25 μm. Data were shown as mean ± SD. EC, endothelial cell; FB, fibroblast; KD, knockdown. See also and .

Article Snippet: The following primary antibodies were used: mouse anti-claudin-5 (1:500, Invitrogen, 35–2500), rabbit anti-ZO-1 (1:500, Thermofisher, 61–7300), rabbit anti-caveolin-1 (1:1000, cell signaling, 3238S), rat anti-meca32 (1:200, Novus, NB100-77668), goat anti-TIMP2 (1:200, R&D, AF971), and mouse anti-actin (1:2000, Sigma, A5441).

Techniques: In Vitro, Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Control, Blocking Assay, Western Blot, Expressing, Transduction, shRNA, Sequencing, MANN-WHITNEY, Knockdown, Saline, Recombinant

Journal: Cell reports

Article Title: Fibroblasts repair blood-brain barrier damage and hemorrhagic brain injury via TIMP2

doi: 10.1016/j.celrep.2022.111709

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following primary antibodies were used: mouse anti-claudin-5 (1:500, Invitrogen, 35–2500), rabbit anti-ZO-1 (1:500, Thermofisher, 61–7300), rabbit anti-caveolin-1 (1:1000, cell signaling, 3238S), rat anti-meca32 (1:200, Novus, NB100-77668), goat anti-TIMP2 (1:200, R&D, AF971), and mouse anti-actin (1:2000, Sigma, A5441).

Techniques: Virus, Recombinant, Avidin-Biotin Assay, RNAscope, In Situ Hybridization, Software