anti epha2 polyclonal goat igg  (R&D Systems)

Journal: Biomedicines

Article Title: Eph/Ephrin Promotes the Adhesion of Liver Tissue-Resident Macrophages to a Mimicked Surface of Liver Sinusoidal Endothelial Cells

doi: 10.3390/biomedicines10123234

Figure Lengend Snippet: EphA2, ephrin-A1, EphB4, and ephrin-B1 localization in LSECs and Kupffer cells. Immunofluorescence micrographs showing EphA2, ephrin-A1, EphB4, and ephrin-B1 immunoreactivity (red) in CD146-positive LSECs (green) and F4/80-positive Kupffer cells (green) in the mouse liver. Cryostat sections were stained with the indicated antibodies and DAPI (blue). Green fluorescence images were merged with red, and/or blue fluorescence images in the same field. EphA2, ephrin-A1, EphB4, and ephrin-B1 immunoreactivity was localized in CD146-positive LSECs and weakly localized in F4/80-positive Kupffer cells (arrows). M, marginal region of the hepatic lobules; Pc, pericentral region of the hepatic lobules.

Article Snippet: Goat polyclonal antibodies against EphA2 (AF639), EphB4 (AF446), and ephrin-B1 (AF473) (R&D Systems, Minneapolis, MN, USA) and rabbit polyclonal antibody against ephrin-A1 (SAB4500696; Sigma-Aldrich) were used.

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Biomedicines

Article Title: Eph/Ephrin Promotes the Adhesion of Liver Tissue-Resident Macrophages to a Mimicked Surface of Liver Sinusoidal Endothelial Cells

doi: 10.3390/biomedicines10123234

Figure Lengend Snippet: Expression of Ephs and ephrins in the mouse liver, liver Mø propagated using mixed culture, and LSECs. ( A ) The protein expression and tyrosine phosphorylation (PTyr) of EphA2 and EphB4 were detected using Western blotting in the mouse liver. EphA2 and EphB4 in the liver are highly and weakly tyrosine-phosphorylated, respectively. IP, immunoprecipitation. ( B , C ) RT-PCR analysis of all Ephs and ephrins in liver Mø propagated using mixed culture ( B ) and primary LSECs ( C ). Liver Mø express Epha2 , Epha4 , Efna1 , Efna4 , Efna5, Ephb3 , Ephb4 , Ephb6 , Efnb1 , Efnb2 , and Efnb3 whereas LSECs express Epha2 , Efna1 , Ephb4 , Efnb1 , Efnb2 , and Efnb3 .

Article Snippet: Goat polyclonal antibodies against EphA2 (AF639), EphB4 (AF446), and ephrin-B1 (AF473) (R&D Systems, Minneapolis, MN, USA) and rabbit polyclonal antibody against ephrin-A1 (SAB4500696; Sigma-Aldrich) were used.

Techniques: Expressing, Phospho-proteomics, Western Blot, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

Journal: JCI Insight

Article Title: MyD88/CD40 signaling retains CAR T cells in a less differentiated state

doi: 10.1172/jci.insight.136093

Figure Lengend Snippet: ( A ) Scheme of EphA2-CAR constructs. ( B ) Summary plot of %tCD19 + T cells ( n = 5, mean ± SEM, 1-way ANOVA with Tukey’s test for multiple comparisons). ( C ) Summary plot of %F(ab′) 2 -positive T cells ( n = 5, mean ± SEM, 1-way ANOVA with Tukey’s test for multiple comparisons). ( D ) CAR T cell production of Th1 (IFN-γ and IL-2) and Th2 (IL-4 and IL-10) cytokines after 24-hour coculture at a 2:1 ratio against EphA2-positive (LM7, U373 WT) and EphA2-negative (BV173, U373 EphA2 KO) cell lines or in media alone ( n = 5, mean ± SEM, 2-way ANOVA with Dunnett’s test for multiple comparisons, all statistical analysis is in comparison with NT cells). Dot colors: black, media; light gray, BV173 (EphA2 negative); dark gray, U373 EphA2 KO (EphA2 negative); dark blue: U373 (EphA2 positive); light blue, LM7 (EphA2 positive). ( E ) Summary plots of Th1 and Th2 cytokine production against EphA2-positive cell lines U373 and LM7 ( n = 5, mean ± SEM, values were log transformed before 2-way ANOVA with Tukey’s test for multiple comparisons). ( F ) CAR T cells were incubated with increasing amounts of tumor cells for 24 hours, and the remaining live tumor cells were quantified with an MTS assay (2-way ANOVA with Tukey’s test for multiple comparisons, mean ± SEM, LM7: n = 4, U373 WT and U373 EphA2 KO: n = 9). For LM7 and U373, asterisks refer to statistical comparison of MC-CAR with NT and CD28-CAR with MC-CAR. For U373 EphA2 KO, asterisks refer to statistical comparison of 41BB-CAR with NT and CD28-CAR with NT. # P < 0.1; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: After SDS-PAGE and wet transfer, the membrane was blocked with 5% milk in TBS-Tween (TBST), then incubated with primary antibodies: goat anti-human EphA2 (R&D Systems, Bio-Techne, AF3035), rabbit anti-human Bcl-2 (clone D55G8, Cell Signaling Technology), rabbit anti-human Myb (clone D2R4Y, Cell Signaling Technology), or mouse anti-human GAPDH (clone 0411, Santa Cruz Biotechnology).

Techniques: Construct, Comparison, Transformation Assay, Incubation, MTS Assay

Journal: JCI Insight

Article Title: MyD88/CD40 signaling retains CAR T cells in a less differentiated state

doi: 10.1172/jci.insight.136093

Figure Lengend Snippet: T cells were cocultured with tumor cells at a 2:1 ratio with weekly restimulation against fresh tumor cells until they lost their effector function and no longer killed all the tumor cells. ( A ) Average expansion of CAR T cells against EphA2-positive (U373 and LM7) and U373 EphA2 KO cell line (mean ± SEM, LM7: n = 4; U373: n = 8 [NT, CD28, MC], n = 4 [41BB]; U373 KO: n = 8 [NT, CD28, MC], n = 6 [41BB]). ( B ) Summary of the maximum expansion CAR T cells from individual donors achieved against EphA2-positive tumor cells and the maximum number of times CAR T cells were able to kill fresh EphA2-positive tumor cells ( n = 12 [NT, CD28, and MC], n = 8 [41BB]; median and quartiles, 1-way ANOVA with Tukey’s test for multiple comparisons). T cells were phenotyped 7 days after stimulation with U373. ( C ) Summary plot of CD4/CD8 composition after stimulation with U373 ( n = 3, mean ± SEM). ( D ) Scheme for phenotyping T cells and representative flow cytometry plots of CCR7 and CD45RA expression on CAR T cells after stimulation with U373. ( E ) Summary plot of T cell phenotype after stimulation with U373 ( n = 4, mean ± SEM, 2-way ANOVA with Tukey’s test for multiple comparisons). All statistical tests were performed in comparison with MC-CAR T cells (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001).

Article Snippet: After SDS-PAGE and wet transfer, the membrane was blocked with 5% milk in TBS-Tween (TBST), then incubated with primary antibodies: goat anti-human EphA2 (R&D Systems, Bio-Techne, AF3035), rabbit anti-human Bcl-2 (clone D55G8, Cell Signaling Technology), rabbit anti-human Myb (clone D2R4Y, Cell Signaling Technology), or mouse anti-human GAPDH (clone 0411, Santa Cruz Biotechnology).

Techniques: Flow Cytometry, Expressing, Comparison

Journal: JCI Insight

Article Title: MyD88/CD40 signaling retains CAR T cells in a less differentiated state

doi: 10.1172/jci.insight.136093

Figure Lengend Snippet: ( A – D ) NSG mice were injected with 1 × 10 6 LM7-ffLuc i.p. One week later, mice were injected with 1 × 10 4 or 1 × 10 5 CD28-, 41BB-, or MC-CAR T cells. PBS and 1 × 10 5 Delta-CAR T cells were used as controls. ( A ) Total flux from tumor cells in all mice treated with 1 × 10 4 EphA2 CAR T cells (PBS: n = 5; CD28, 41BB, MC: n = 10). ( B ) Total flux from tumor cells in all mice treated with 1 × 10 5 EphA2 CAR T cells (Delta: n = 5, CD28: n = 8, 41BB: n = 9, MC: n = 10). ( C and D ) Kaplan-Meier survival analysis of mice treated with 1 × 10 4 ( C ) or 1 × 10 5 ( D ) EphA2 CAR T cells (log-rank Mantel-Cox test with Bonferroni’s correction for multiple comparisons; * P < 0.05; ** P < 0.01; *** P < 0.001). Experiments were repeated twice with CAR T cells generated from 2 different healthy donors.

Article Snippet: After SDS-PAGE and wet transfer, the membrane was blocked with 5% milk in TBS-Tween (TBST), then incubated with primary antibodies: goat anti-human EphA2 (R&D Systems, Bio-Techne, AF3035), rabbit anti-human Bcl-2 (clone D55G8, Cell Signaling Technology), rabbit anti-human Myb (clone D2R4Y, Cell Signaling Technology), or mouse anti-human GAPDH (clone 0411, Santa Cruz Biotechnology).

Techniques: Injection, Generated