Journal: Cell reports

Article Title: SerpinB3 drives cancer stem cell survival in glioblastoma

doi: 10.1016/j.celrep.2022.111348

Figure Lengend Snippet: (A) Graphical abstract of His-JAM-A pulldown and liquid chromatography-mass spectrometry (LC-MS) procedure. (B) Verification of LC-MS results by western blot. His-tagged JAM-A was overexpressed in T4121 cancer stem cells (CSCs), and protein was isolated and mixed with nickel beads. The bound fraction was subjected to immunoblotting with antibodies to SerpinB3 and JAM-A. (C) Immunofluorescent staining demonstrating co-expression of JAM-A and SerpinB3 in T387 PDX glioblastoma tumor model (scale bar, 10 μm). (D) JAM-A was knocked down in T4121 CSCs with 2 separate shRNA constructs, and SerpinB3 expression was measured. Actin was used as a loading control in this and all subsequent western blots. (E) T387 and T4121 CSCs expressing JAM-A KD2 shRNA or NT control were treated with cycloheximide, and SerpinB3 expression was measured at 6 and 12 h post-treatment. (F) Western blot demonstrating knockdown of SerpinB3 with each shRNA, KD1, and KD2. (G) Fold change in cell viability at day 7, normalized to day 0, in 3 PDX glioblastoma models. Cell viability measured with CellTiter-Glo Luminescent Cell Viability Assay (5 technical replicates per condition, per tumor model). (H and I) Kaplan-Meier curves depicting survival of mice with 20,000 T4121 or T387 tumor cells intracranially injected. Cells were transfected with either non-target (SHC002) or SerpinB3 (KD1 or KD2) shRNA, with n = 10 mice per group. p < 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001, as determined by 1-way ANOVA with Dunnett’s multiple comparisons test or log rank test for survival data. Error bars represent standard deviations.

Article Snippet: N-terminal His-tagged full-length JAM-A , Sinobiologicals , HG10198-NH.

Techniques: Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Western Blot, Isolation, Staining, Expressing, shRNA, Construct, Cell Viability Assay, Injection, Transfection

Journal: Molecular Biology of the Cell

Article Title: Role of JAM-A tyrosine phosphorylation in epithelial barrier dysfunction during intestinal inflammation

doi: 10.1091/mbc.e18-08-0531

Figure Lengend Snippet: Figure 1 JAM-A is tyrosine phosphorylated at Y280. a: Human JAM-A highlighting

Article Snippet: Rabbit anti-p-JAM-A Y280 was from Rockland Inc., Limerick, PA, catalog: 600-401-GN5.

Techniques:

Journal: Molecular Biology of the Cell

Article Title: Role of JAM-A tyrosine phosphorylation in epithelial barrier dysfunction during intestinal inflammation

doi: 10.1091/mbc.e18-08-0531

Figure Lengend Snippet: Figure 3 Yes-1 regulates phosphorylation of JAM-A Y280. a: The Src family kinase inhibitor PP2 (1 M) inhibits pervanadate-dependent phosphorylation of JAM-A Y280. Immunofluorescence staining of p-JAM-A Y280 and total JAM-A in T84 monolayers pre- treated with/without PP2 followed by exposure to pervanadate. Similar results were observed in SK CO-15 cells (not shown). b: Dose-dependent inhibition of p-JAM-A Y280 by PP2 after pervanadate treatment. T84 cells were lysed after treatment with increasing concentrations of PP2 followed by exposure to pervanadate. Lysates were then immunoblotted for p-JAM-A Y280, total JAM-A, and total Src. P-EGFR Y1068 was used as a positive control for PP2. Calnexin was used as a loading control. c: Co- immunoprecipitation of Yes1/Lyn with JAM-A. SK CO-15 cells stably expressing HA- tagged full-length JAM-A were lysed with 1% Triton X-100 lysis buffer followed by

Article Snippet: Rabbit anti-p-JAM-A Y280 was from Rockland Inc., Limerick, PA, catalog: 600-401-GN5.

Techniques: Phospho-proteomics, Immunofluorescence, Staining, Inhibition, Positive Control, Control, Immunoprecipitation, Stable Transfection, Expressing, Lysis

Journal: Molecular Biology of the Cell

Article Title: Role of JAM-A tyrosine phosphorylation in epithelial barrier dysfunction during intestinal inflammation

doi: 10.1091/mbc.e18-08-0531

Figure Lengend Snippet: Figure 4. PTPN13 acts as a phosphatase for p-JAM-A Y280.

Article Snippet: Rabbit anti-p-JAM-A Y280 was from Rockland Inc., Limerick, PA, catalog: 600-401-GN5.

Techniques:

Journal: Molecular Biology of the Cell

Article Title: Role of JAM-A tyrosine phosphorylation in epithelial barrier dysfunction during intestinal inflammation

doi: 10.1091/mbc.e18-08-0531

Figure Lengend Snippet: Figure 5. Phosphorylation of JAM-A Y280 results in decreased JAM-A association

Article Snippet: Rabbit anti-p-JAM-A Y280 was from Rockland Inc., Limerick, PA, catalog: 600-401-GN5.

Techniques: Phospho-proteomics

Journal: Molecular Biology of the Cell

Article Title: Role of JAM-A tyrosine phosphorylation in epithelial barrier dysfunction during intestinal inflammation

doi: 10.1091/mbc.e18-08-0531

Figure Lengend Snippet: Figure 6 p-JAM-A Y280 is increased in mouse models of experimental intestinal

Article Snippet: Rabbit anti-p-JAM-A Y280 was from Rockland Inc., Limerick, PA, catalog: 600-401-GN5.

Techniques: