Journal: Journal of the American Society of Nephrology

Article Title: Negative Modulation of B Cell Activation by Melanocortin 1 Receptor Signaling Protects against Membranous Nephropathy

doi: 10.1681/asn.2022050605

Figure Lengend Snippet: Figure 8. Loss of MC1R potentiates B-cell activation and en- hances autologous humoral immune responses in rats with PHN. (A) Animals were treated as elaborated in Figure 1F. Splenocytes derived from WT or KO rats with PHN were pro- cessed for flow cytometry analysis of CD1381 cells. (B) Arbitrary levels of sheep IgG-specific rat IgG in sera collected from WT or KO rats with PHN, as estimated by ELISA of diluted sera; aP,0.05 versus WT rats with PHN (n56). (C) Schematic diagram depicting the working model for negative regulation of B-cell activation by MC1R-mediated melanocortinergic signaling. As a typical G-protein coupled receptor, MC1R is expressed by B lympho- cytes and is able to trigger the cAMP signaling pathway on ac- tivation by diverse melanocortins, such as pan-MCR agonists like ACTH and NDP-MSH, or the selective MC1R agonist MS05. Subsequently, cAMP signaling downstream of MC1R activates MITF, one of the major signaling transducers of MC1R pathway and a critical regulator of a plethora of key cellular processes, including melanogenesis in melanocytes and cellular differenti- ation in nonmelanocytic cells. In B lymphocytes, MITF acts as a repressor of IRF4, a characteristic lymphoid transcription factor essential for B-cell development and maturation. MC1R-medi- ated melanocortinergic signaling suppresses B-cell activation and plasmacytic differentiation and reduces IgG production via the cAMP/MITF/IRF4 pathway, which could be intercepted by SQ22536, an adenylyl cyclase inhibitor. This MC1R-mediated B- cell regulatory signaling is defective in B cells with MC1R KO and could be partially restored by the cAMP mimetic dBcAMP.

Article Snippet: IgG levels in cell culture supernatants were detected by using a rat IgG ELISA kit (Aviva Systems Biology, San Diego, CA) according to the manufacturer’s instruction.

Techniques: Activation Assay, Derivative Assay, Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 1. PDL1 expression in cancer and noncancer cell lines. A, relative expression of PDL1 from total RNA isolated from cell lines. B, representative PDL1 protein expression levels in corresponding high- and low- expressing cell lines by IHC. C, representative PDL1 protein expression levels in corresponding high- and low- expressing cell lines by flow cytometry.

Article Snippet: Unfortunately, the ProSci rabbit polyclonal failed to detect PDL1 on the positive control cell line SKOV3, and the XW mouse mAb exhibited unspecific binding on the negative control cell line MCF7 (Supplementary Fig. S1).

Techniques: Expressing, Isolation, Cytometry

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 2. Relative expression of PDL1 in 38 osteosarcoma specimens. PDL1 expression was evaluated from total RNA by quantitative real-time RT-PCR and showed that the expression levels range over 4 log (5,000-fold).

Article Snippet: Unfortunately, the ProSci rabbit polyclonal failed to detect PDL1 on the positive control cell line SKOV3, and the XW mouse mAb exhibited unspecific binding on the negative control cell line MCF7 (Supplementary Fig. S1).

Techniques: Expressing, Quantitative RT-PCR

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 3. Overall survival of 37 patients with osteosarcoma in relation to PDL1 gene expression. The median overall survival for PDL1-low patients was 89 months compared with 28 months for PDL1-high patients, which showed a trend but was not statistically significant (P ¼ 0.0544).

Article Snippet: Unfortunately, the ProSci rabbit polyclonal failed to detect PDL1 on the positive control cell line SKOV3, and the XW mouse mAb exhibited unspecific binding on the negative control cell line MCF7 (Supplementary Fig. S1).

Techniques: Gene Expression

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 4. Correlation between PDL1 gene expression and TILs by IHC. A, representative TILs in osteosarcoma tissues (400); score 0, no TILs; 1, rare/few TILs; 2, brisk/prominent TILs. B, significant positive correlation was shown between PDL1 gene expression and TILs in patients with osteosarcoma (P ¼ 0.0117).

Article Snippet: Unfortunately, the ProSci rabbit polyclonal failed to detect PDL1 on the positive control cell line SKOV3, and the XW mouse mAb exhibited unspecific binding on the negative control cell line MCF7 (Supplementary Fig. S1).

Techniques: Gene Expression

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 5. Characterization of the origins of metastases. A, PDL1 expression is significantly higher in metastatic osteosarcoma tumors that originate from lung than from other locations (P ¼ 0.0024). B, TILs also exhibit a positive correlation with pulmonary osteosarcoma metastasis compared with nonpulmonary metastasis (P ¼ 0.0443).

Article Snippet: Unfortunately, the ProSci rabbit polyclonal failed to detect PDL1 on the positive control cell line SKOV3, and the XW mouse mAb exhibited unspecific binding on the negative control cell line MCF7 (Supplementary Fig. S1).

Techniques: Expressing

Journal: Clinical and Experimental Immunology

Article Title: The effects of maternal anti-alpha-enolase antibody expression on the brain development in offspring

doi: 10.1093/cei/uxac086

Figure Lengend Snippet: the levels of ENO1Ab in the H-ENO1Ab dams and their pups. Serum and amniotic fluid samples were collected as depicted in . Anti-ENO1 total IgG and its subtypes were measured by enzyme-linked immunosorbent assay (ELISA). The results are shown above for the dams (A, n = 6-14/group, ** P < 0.01, *** P < 0.001 vs. the control group) and the pups (B, n = 14-19 for total IgG, n = 5-8 for IgG1, n = 5-9 for IgG2a, IgG2b and IgG3, ** P < 0.01, *** P < 0.001, not significantly (NS) vs. the pups of the control group).

Article Snippet: After 2 h incubation at RT followed by repeated washing, the plates were further incubated with diluted horseradish peroxidase (HRP)-labeled goat anti-mouse total IgG (1:200 diluted for TgAb;1:10 000 for ENO1Ab), IgG1(1:1000 for ENO1Ab), IgG2a (1:1000 for ENO1Ab), IgG2b (1:1000 for ENO1Ab), or IgG3 antibodies (1:1000 for ENO1Ab), which were all from Bethyl (USA).

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Clinical and Experimental Immunology

Article Title: The effects of maternal anti-alpha-enolase antibody expression on the brain development in offspring

doi: 10.1093/cei/uxac086

Figure Lengend Snippet: IgG deposition and the distribution of C3, MAC, and CD16 in the brain tissue of the pups from H-ENO1Ab dams. All the pups were obtained as depicted in . (A) Representative images of IgG deposition in the brain tissues from the pups of H-ENO1Ab dams and those of the control group on P10 and P40 ( n = 4/group) after immunofluorescence (IF) staining with FITC-conjugated goat anti-mouse IgG (green, ×200). (B) Representative images for the colocalization of IgG and ENO1, NeuN, and CD34 in the brain tissues from the pups of H-ENO1Ab dams and those of the control group on P10 ( n = 4/group) after double IF staining with FITC-conjugated goat anti-mouse IgG (green) and rabbit anti-ENO1 and NeuN, rat anti-CD34 (red, ×200). (C and E) Representative images of the colocalization of IgG and C3 and CD16 in the brain tissues from the pups of H-ENO1Ab dams and those of the control group on P10 and P40 ( n = 4/group) after double IF staining with FITC-conjugated goat anti-mouse IgG (green) and rabbit anti-C3 and CD16 (red, ×200). (D) Representative images of MAC expression in the brain tissues from the pups of H-ENO1Ab dams and the those of the control group on P10 and P40 ( n = 4/group) after IF staining with mouse anti-C5b-9 (green, ×200). Nuclei were counterstained with DAPI (blue).

Article Snippet: After 2 h incubation at RT followed by repeated washing, the plates were further incubated with diluted horseradish peroxidase (HRP)-labeled goat anti-mouse total IgG (1:200 diluted for TgAb;1:10 000 for ENO1Ab), IgG1(1:1000 for ENO1Ab), IgG2a (1:1000 for ENO1Ab), IgG2b (1:1000 for ENO1Ab), or IgG3 antibodies (1:1000 for ENO1Ab), which were all from Bethyl (USA).

Techniques: Control, Immunofluorescence, Staining, Expressing