Journal: Journal of leukocyte biology

Article Title: K48- and K27-mutant ubiquitin regulates adaptive immune response by affecting cross-presentation in bone marrow precursor cells.

doi: 10.1002/JLB.4MA0222-419RR

Figure Lengend Snippet: FIGURE 2 The treatment with MG132 decreases the cytolytic activity of bone marrow precursor cell -activated T cells. (A–E) Murine BMPC was pretreated with MG132 at indicated concentration prior to 5–6 h OVA (100 μg/ml) incubation. Then, 1 × 105 BMPC was intraperitoneally transferred into C57BL/6 recipients. 5–7 d after adoptive transfer, the splenocyte of the recipients was prepared. The expressions of perforin (A and C) and granzyme B (B and D) were determined by Western blot (A and B) and flow cytometric analyses with intercellular staining (C and D). The cytolytic activity of BMPC-activated T cells was assessed by incubating effector cells with CFSE-labeled E.G7-OVA target cells (tumor cells with specific expression of OVA) (E). CFSE value of the cell was analyzed by flow cytometry, and the cytolytic activity was calculated (E). For Western blot analyses, β-actin was used as an internal control. One representative from 3 independent experiments is shown. Data were presented as the mean ± SEM, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, one-way or two-way ANOVA with Newman–Keuls posttest

Article Snippet: Antibodies to β-actin (13E5, #4970), IFN-γ (D3H2, #8455), perforin (E7D8R, #62550), and HRP-conjugated secondary antibody were bought from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activity Assay, Concentration Assay, Incubation, Adoptive Transfer Assay, Western Blot, Staining, Labeling, Expressing, Flow Cytometry, Control

Journal: Journal of leukocyte biology

Article Title: K48- and K27-mutant ubiquitin regulates adaptive immune response by affecting cross-presentation in bone marrow precursor cells.

doi: 10.1002/JLB.4MA0222-419RR

Figure Lengend Snippet: FIGURE 4 The deficiency of ubiquitin attenuates the cytolytic activity of bone marrow precursor cell-activated T cells. (A–E) Murine ubiquitin deficient and control BMPC were conferred 5–6 h OVA (100 μg/ml) incubation. Then, 1 × 105 DC was intraperitoneally transferred into C57BL/6 recipients. 5–7 d after adoptive transfer, the splenocytes of the recipients was prepared. The expressions of perforin (A and C) and granzyme B (B and D) were determined by Western blot (A and B) and flow cytometric analyses with intercellular staining (C and D). The cytolytic activity of BMPC-activated T cells was assessed by incubating effector cells with CFSE-labeled E.G7-OVA target cells (tumor cells with specific expression of OVA) (E). CFSE value of the cell was determined by flow cytometry, and the cytolytic activity was calculated (E). For Western blot analyses, β-actin was used as an internal control. One representative from 3 independent experiments is shown. Data were presented as the mean ± SEM, n = 3. **p < 0.01, ***p < 0.001, one-way or two-way ANOVA with Newman–Keuls posttest. Ub, ubiquitin; si, siRNA

Article Snippet: Antibodies to β-actin (13E5, #4970), IFN-γ (D3H2, #8455), perforin (E7D8R, #62550), and HRP-conjugated secondary antibody were bought from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Ubiquitin Proteomics, Activity Assay, Control, Incubation, Adoptive Transfer Assay, Western Blot, Staining, Labeling, Expressing, Flow Cytometry

Journal: Journal of leukocyte biology

Article Title: K48- and K27-mutant ubiquitin regulates adaptive immune response by affecting cross-presentation in bone marrow precursor cells.

doi: 10.1002/JLB.4MA0222-419RR

Figure Lengend Snippet: FIGURE 6 K48-mutant ubiquitin decreases the cytolytic activity of bone marrow precursor cell-activated T cells. (A–E) Murine ubiquitin deficient and control BMPC were incubated with OVA (100 μg/ml) for 5–6 h in the presence of K48-mutant ubiquitin (K48R) at the final concentration of 3 × 10−5 mol/L. 1 × 105 BMPC was intraperitoneally transferred into C57BL/6 recipients. 5–7 d after adoptive transfer, the splenocyte of the recipients was prepared. The expressions of perforin (A and C) and granzyme B (B and D) were determined by Western blot (A and B) and flow cytometric analyses with intercellular staining (C and D). The cytolytic activity of BMPC-activated T cells was assessed by incubating effector cells with CFSE-labeled E.G7-OVA target cells (tumor cells with specific expression of OVA) (E). CFSE value of the cell was analyzed by flow cytometry, and the cytolytic activity was calculated (E). For Western blot analyses, β-actin was used as an internal control. One representative from 3 independent experiments is shown. Data were presented as the mean ± SEM, n = 3. **p < 0.01, ***p < 0.001, one-way or two-way ANOVA with Newman–Keuls posttest. si, siRNA; Ub, ubiquitin

Article Snippet: Antibodies to β-actin (13E5, #4970), IFN-γ (D3H2, #8455), perforin (E7D8R, #62550), and HRP-conjugated secondary antibody were bought from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Mutagenesis, Ubiquitin Proteomics, Activity Assay, Control, Incubation, Concentration Assay, Adoptive Transfer Assay, Western Blot, Staining, Labeling, Expressing, Flow Cytometry

Journal: Journal of leukocyte biology

Article Title: K48- and K27-mutant ubiquitin regulates adaptive immune response by affecting cross-presentation in bone marrow precursor cells.

doi: 10.1002/JLB.4MA0222-419RR

Figure Lengend Snippet: FIGURE 8 K27-mutant ubiquitin decreases the cytolytic activity of bone marrow precursor cell-activated T cells. (A–E) Murine ubiquitin deficient and control BMPC were incubated with OVA (100 μg/ml) for 5–6 h in the presence of K27-mutant ubiquitin (K27R) at the final concentration of 3 × 10−5 mol/L. 1 × 105 BMPC was intraperitoneally transferred into C57BL/6 recipients. 5–7 d after adoptive transfer, the splenocyte of the recipients was prepared. The expressions of perforin (A and C) and granzyme B (B and D) were determined by Western blot (A and B) and flow cytometric analyses with intercellular staining (C and D). The cytolytic activity of BMPC-activated T cells was assessed by incubating effector cells with CFSE-labeled E.G7-OVA target cells (tumor cells with specific expression of OVA) (E). CFSE value of the cell was analyzed by flow cytometry, and the cytolytic activity was calculated (E). For Western blot analyses, β-actin was used as an internal control. One representative from 3 independent experiments is shown. Data were presented as the mean ± SEM, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, one-way or two-way ANOVA with Newman–Keuls posttest. si, siRNA; Ub, ubiquitin

Article Snippet: Antibodies to β-actin (13E5, #4970), IFN-γ (D3H2, #8455), perforin (E7D8R, #62550), and HRP-conjugated secondary antibody were bought from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Mutagenesis, Ubiquitin Proteomics, Activity Assay, Control, Incubation, Concentration Assay, Adoptive Transfer Assay, Western Blot, Staining, Labeling, Expressing, Flow Cytometry

Journal: Cell death & disease

Article Title: Transcription factor activating enhancer-binding protein 2ε (AP2ε) modulates phenotypic plasticity and progression of malignant melanoma.

doi: 10.1038/s41419-024-06733-3

Figure Lengend Snippet: Fig. 5 AP2ε regulates phenotypic plasticity of melanoma cells. A Representative Western Blot images depicting murine MITF and BRN2 protein levels in primary AP2ε-/-/Tg(GRM1) and Tg(GRM1) cells (left panels) and human MITF and BRN2 in Mel Juso cells transfected with the AP2ε-overexpression (hAP2ε) plasmid and control vector pCMX (right panels). β-Actin served as loading control. B Immunohistochemical staining of MITF and BRN2 protein expression in Mel Im melanoma cells bioprinted in CIB (Magnification: 20x). Quantification of percentage of cells with “no”, “weak”, or “strong” MITF and BRN2 expression. Data are represented as mean ± SEM; *p < 0.05; ns: not significant (Two-way- ANOVA with Fisher’s LSD test). C mRNA- and protein expression analysis for Snail1 in AP2ε-/-/Tg(GRM1) and Tg(GRM1) cells (n = 3). D mRNA- and protein expression analysis for Snail1 in Mel Juso cells transfected with the AP2ε-overexpression (hAP2ε) plasmid and control vector pCMX (n = 3). E mRNA- and protein expression analysis for E-cadherin in AP2ε-/-/Tg(GRM1) and Tg(GRM1) cells (n = 3). F mRNA- and protein expression analysis for E-cadherin in Mel Juso cells transfected with the AP2ε-overexpression (hAP2ε) plasmid and control vector pCMX (n = 3). Data information: All data from at least three independent experiments are represented as mean ± SEM; *p < 0.05 (Two-tailed Student’s t-test; ns: not significant).

Article Snippet: Primary antibodies against MITF (1:500 in 5% BSA, Santa Cruz, sc-515925, RRID:AB_2828036), BRN2 (1:500 in 5% BSA/1x TBS-T, Santa Cruz, sc393324, RRID:AB_2737347), Snail1 (1:1,000 in 5% BSA/1x TBS-T, Cell Signaling Technology Cat# 3879, RRID:AB_2255011) and E-cadherin (1:1,000 in 5% BSA/1x TBS-T, Cell Signaling Technology Cat# 3195, RRID:AB_2291471) were incubated overnight at 4 °C.

Techniques: Western Blot, Transfection, Over Expression, Plasmid Preparation, Control, Immunohistochemical staining, Staining, Expressing, Two Tailed Test