primary mouse antibody to azgp1  (Santa Cruz Biotechnology)

Journal: EMBO Molecular Medicine

Article Title: Loss of EHMT2 enhances NK cell-driven anti-tumor immunity through TGF-β1 suppression

doi: 10.1038/s44321-025-00357-6

Figure Lengend Snippet: ( A ) Mel92.1 and MP41 cells expressing either nonspecific single-guide RNA (NS sgRNA) or EHMT2 sgRNAs were analyzed for the indicated proteins using immunoblotting. Histone H3 and ACTINB were used as loading controls. ( B ) Mel92.1 and MP41 cells expressing either NS sgRNA or EHMT2 sgRNAs were analyzed for NK cell-mediated cytotoxicity using an LDH-based cytotoxicity assay. Relative NK cell-mediated cytotoxicity under indicated conditions are plotted. ( n = 6). P values were calculated using unpaired two-tailed Student’s t -test. ( C ) Mel92.1 and MP41 cells expressing either nonspecific (NS) shRNA or AZGP1 shRNAs were analyzed for mRNA expression for AZGP1 mRNA were analyzed using RT-qPCR analysis. Relative mRNA expression for indicated genes are plotted. ACTINB was used for normalization. ( n = 3). P values were calculated using unpaired two-tailed Student’s t -test. ( D ) Mel92.1 and MP41 cells expressing either NS shRNA or AZGP1 shRNAs were analyzed for the expression of AZGP1 by immunoblotting. ACTINB was used as a loading control. ( E ) Mel92.1 cells expressing nonspecific NS shRNA, AZGP1 shRNA, TGF-β1 shRNA or both AZGP1 shRNA and TGF-β1 shRNA were analyzed by immunoblotting for the indicated proteins. ACTINB was used as a loading control. ( F ) Mel92.1 cells expressing either NS shRNA, AZGP1 shRNA, TGF-β1 shRNA or both AZGP1 shRNA and TGF-β1 shRNA were treated with DMSO or EHMT2 inhibitor UNC0642 (1 μM) for 48 h and were analyzed for NK cell-mediated cytotoxicity using an LDH-based cytotoxicity assay. Relative NK cell-mediated cytotoxicity under the indicated conditions is plotted. ( n = 6). P values were calculated using unpaired two-tailed Student’s t -test. All quantitative data were presented as the mean ± SEM.

Article Snippet: AZGP1 (Diluted at 1:500 for WB) , Proteintech , 66178-1-Ig RRID: AB_2881573.

Techniques: Expressing, Western Blot, LDH Cytotoxicity Assay, Two Tailed Test, shRNA, Quantitative RT-PCR, Control

Journal: EMBO Molecular Medicine

Article Title: Loss of EHMT2 enhances NK cell-driven anti-tumor immunity through TGF-β1 suppression

doi: 10.1038/s44321-025-00357-6

Figure Lengend Snippet: ( A ) Heatmap showing differentially expressed mRNAs in Mel92.1 and MP41 cells following 48 h treatment with UNC0642 (1 μM) or A366 (1 μM) compared to DMSO-treated cells. ( B ) Heatmap showing commonly upregulated mRNAs in Mel92.1 and MP41 cells following 48 h treatment with UNC0642 (1 μM) and A366 (1 μM) compared to DMSO-treated cells. ( C ) Mel92.1 cells treated with DMSO or EHMT2 inhibitors UNC0642 (1 μM) or A366 (1 μM) for 48 h were analyzed for the indicated mRNAs using RT-qPCR. mRNA expression relative to DMSO-treated cells is plotted. ACTINB was used for normalization. ( n = 3). P values were calculated using an unpaired two-tailed Student’s t -test. ( D ) MP41 cells treated with DMSO or EHMT2 inhibitors UNC0642 (1 μM) or A366 (1 μM) for 48 h were analyzed for the indicated mRNA using RT-qPCR. mRNA expression relative to DMSO-treated cells is plotted. ACTINB was used for normalization. ( n = 3). P values were calculated using an unpaired two-tailed Student’s t -test. ( E ) Immunoblotting for AZGP1 in Mel92.1 and MP41 cells treated with DMSO or EHMT2 inhibitors UNC0642 (1 μM) or A366 (1 μM) for 48 h. ACTINB was used as a loading control. ( F ) EHMT2 enrichment on the AZGP1 or ACTINB promoters was measured in Mel92.1 cells using the CUT&RUN assay. Relative fold enrichment of EHMT2 on the AZGP1 and ACTINB promoters compared to the IgG control is plotted. ( n = 3). P values were calculated using an unpaired two-tailed Student’s t -test. ( G ) H3K9me2 mark enrichment on the AZGP1 or ACTINB promoters was measured in Mel92.1 cells using CUT&RUN assays. Relative fold enrichment of the H3K9me2 mark on the AZGP1 and ACTINB promoters compared to IgG control is plotted. ( n = 3). P values were calculated using an unpaired two-tailed Student’s t -test. ( H ) Overall survival for UM patients with higher ( n = 40) or lower ( n = 40) levels of AZGP1 mRNA expression in the UM TCGA dataset. The log-rank test was used to calculate the p value of comparing Kaplan–Meier curves, and the Wald test was used to calculate the p value of Cox proportional hazards regression analysis. ( I ) Mel92.1 and MP41 cells expressing either an empty vector or V5-tagged AZGP1 ORF were analyzed for the expression of AZGP1 by immunoblotting using V5-tag antibody or AZGP1 antibody. ACTINB was used as a loading control. ( J ). Mel92.1 and MP41 cells expressing either an empty vector or V5-tagged AZGP 1 ORF were analyzed for NK cell-mediated cytotoxicity using an LDH-based cytotoxicity assay. Relative NK cell-mediated cytotoxicity under the indicated conditions are plotted. ( n = 6). P values were calculated using an unpaired two-tailed Student’s t -test. ( K ) Mel92.1 and MP41 cells expressing either nonspecific (NS) shRNA or AZGP1 shRNAs were treated with DMSO or EHMT2 inhibitors UNC0642 (1 μM) for 48 h and analyzed for NK cell-mediated cytotoxicity using LDH-based cytotoxicity assay. Relative NK cell-mediated cytotoxicity under the indicated conditions are plotted. ( n = 5 for Mel92.1 and n = 6 for MP41). P values were calculated using unpaired two-tailed Student’s t -test. All quantitative data were presented as mean ± SEM. .

Article Snippet: AZGP1 (Diluted at 1:500 for WB) , Proteintech , 66178-1-Ig RRID: AB_2881573.

Techniques: Quantitative RT-PCR, Expressing, Two Tailed Test, Western Blot, Control, Plasmid Preparation, LDH Cytotoxicity Assay, shRNA

Journal: EMBO Molecular Medicine

Article Title: Loss of EHMT2 enhances NK cell-driven anti-tumor immunity through TGF-β1 suppression

doi: 10.1038/s44321-025-00357-6

Figure Lengend Snippet: ( A ) Immunoblotting for measuring expression of the indicated proteins in Mel92.1 and MP41 cells treated with either DMSO or EHMT2 inhibitors UNC0642 (1 μM) or A366 (1 μM) for 48 h. Histone H3 and ACTINB were used as loading controls. ( B ) Immunoblotting for measuring the expression of TGF-β1 in Mel92.1 and MP41 cells expressing either NS shRNA or EHMT2 shRNAs. ACTINB was used as a loading control. ( C ) Immunoblotting for measuring the expression of indicated proteins in Mel92.1 and MP41 cells expressing either an empty vector control or V5-tagged AZGP1 ORF. ACTINB was used as a loading control. ( D ) Immunoblotting for measuring expression of TGF-β1 in Mel92.1 and MP41 cells expressing either NS shRNA or TGF-β1 shRNAs. ACTINB was used as a loading control. ( E ) Mel92.1 and MP41 cells expressing either NS shRNA or TGF-β1 shRNAs were analyzed for NK cell-mediated cytotoxicity using an LDH-based cytotoxicity assay. Relative NK cell-mediated cytotoxicity under the indicated conditions are plotted. ( n = 6). P values were calculated using unpaired two-tailed Student’s t -test. ( F ) Mel92.1 cells expressing either an empty vector or V5-tagged TGF-β1 ORF were analyzed for the expression of TGF-β1 by immunoblotting using V5-tag antibody. ACTINB was used as a loading control. ( G ) Mel92.1 cells expressing either an empty vector or V5-tagged TGF-β1 ORF were treated with DMSO or EHMT2 inhibitor UNC0642 (1 μM) or A366 for 48 h and analyzed for NK cell-mediated cytotoxicity using LDH-based cytotoxicity assay. Relative NK cell-mediated cytotoxicity at the indicated conditions is plotted. ( n = 6). P values were calculated using unpaired two-tailed Student’s t -test. ( H ) MP41 cells expressing either an empty vector or TGF-β1 ORF were analyzed for the expression of TGF-β1 by immunoblotting using V5-tag antibody. ACTINB was used as a loading control. ( I ) MP41 cells expressing either an empty vector or V5-tagged TGF-β1 ORF were treated with DMSO or EHMT2 inhibitors UNC0642 (1 μM) or A366 (1 μM) for 48 h and analyzed for NK cell-mediated cytotoxicity using LDH-based cytotoxicity assay. Relative NK cell-mediated cytotoxicity at the indicated conditions is plotted. ( n = 6). P values were calculated using unpaired two-tailed Student’s t -test. ( J ) Mel92.1 cells treated with DMSO or EHMT2 inhibitor (UNC0642, 1 μM) in Opti-MEM for 48 h. Following this, the conditioned media was collected, concentrated and used to treat NK cells for 24 h. NK cells were analyzed for cytotoxicity against Mel92.1 cells using an LDH-based cytotoxicity assay. Relative NK cell-mediated cytotoxicity at the indicated conditions are plotted. ( n = 6). P values were calculated using unpaired two-tailed Student’s t -test. ( K ) Mel92.1 cells expressing either an empty vector, V5-tagged AZGP1 ORF or V5-tagged TGF-β1 ORF were grown in Opti-MEM for 48 h. Following this, the conditioned media was collected, concentrated and used to treat NK cells for 24 h. NK cells were analyzed for cytotoxicity against Mel92.1 cells using an LDH-based cytotoxicity assay. Relative NK cell-mediated cytotoxicity at the indicated conditions is plotted. ( n = 5). P values were calculated using unpaired two-tailed Student’s t -test. ( L ) Mel92.1 or MP41 cells were grown in Opti-MEM for 48 h. Following this, the conditioned media was either incubated with control IgG antibody or TGF-β1-neutralizing antibody and used to treat NK cells for 24 h. Following this, NK cell-mediated cytotoxicity was measured against Mel92.1 or MP41 cells using an LDH-based cytotoxicity assay. Relative NK cell-mediated cytotoxicity under the indicated conditions are shown. ( n = 6). P values were calculated using unpaired two-tailed Student’s t -test. All quantitative data are shown as the mean ± SEM. .

Article Snippet: AZGP1 (Diluted at 1:500 for WB) , Proteintech , 66178-1-Ig RRID: AB_2881573.

Techniques: Western Blot, Expressing, shRNA, Control, Plasmid Preparation, LDH Cytotoxicity Assay, Two Tailed Test, Incubation

Journal: EMBO Molecular Medicine

Article Title: Loss of EHMT2 enhances NK cell-driven anti-tumor immunity through TGF-β1 suppression

doi: 10.1038/s44321-025-00357-6

Figure Lengend Snippet: ( A ) Mel92.1 and MP41 cells were treated with DMSO or BRD4770 (5 μM) for 48 h and NK cell-mediated cytotoxicity was measured using an LDH-based cytotoxicity assay. Relative NK cell-mediated cytotoxicity under the indicated conditions for indicated UM cell lines is plotted. ( n = 5). P values were calculated using unpaired two-tailed Student’s t -test. ( B ) Mel92.1 and MP41 cells treated with DMSO or BRD4770 (5 μM) for 48 h and were analyzed for AZGP1 mRNA using RT-qPCR. mRNA expression relative to DMSO-treated cells is plotted. ACTINB was used for normalization. (n = 3). P values were calculated using unpaired two-tailed Student’s t -test. ( C ) Mel92.1 and MP41 cells were treated with DMSO or BRD4770 (5 μM) for 48 h and were analyzed for AZGP1 expression by immunoblotting. ACTINB was used as a loading control. ( D ) Mel92.1 and MP41 cells were treated with DMSO or BRD4770 (5 μM) for 48 h and were analyzed for TGF-β1 mRNA using RT-qPCR. mRNA expression relative to DMSO-treated cells is plotted. ACTINB was used for normalization. ( n = 3). P values were calculated using unpaired two-tailed Student’s t -test. ( E ) Mel92.1 and MP41 cells were treated with DMSO or BRD4770 (5 μM) for 48 h and were analyzed for TGF-β1 expression by immunoblotting. ACTINB was used as a loading control. ( F ) Mel92.1 and MP41 cells were treated with DMSO or BRD4770 (5 μM) for 48 h and were analyzed for MICB mRNA using RT-qPCR. mRNA expression relative to DMSO-treated cells is plotted. ACTINB was used for normalization. ( n = 3). P values were calculated using unpaired two-tailed Student’s t -test. ( G ) Mel92.1 and MP41 cells were treated with DMSO or BRD4770 (5 μM) for 48 h and were analyzed for MICB expression by immunoblotting. ACTINB was used as a loading control. ( H ) Mel92.1 and MP41 cells were treated with DMSO or BRD4770 (5 μM) for 48 h and were analyzed for CXCL10 and CCL27 mRNAs using RT-qPCR. mRNA expression relative to DMSO -treated cells is plotted. ACTINB was used for normalization. ( n = 3). P values were calculated using unpaired two-tailed Student’s t -test. All quantitative data were shown as the mean ± SEM.

Article Snippet: AZGP1 (Diluted at 1:500 for WB) , Proteintech , 66178-1-Ig RRID: AB_2881573.

Techniques: LDH Cytotoxicity Assay, Two Tailed Test, Quantitative RT-PCR, Expressing, Western Blot, Control

Journal: Molecular Medicine

Article Title: Secreted AZGP1 induced by 5-FU binds to PD-L1 and promotes apoptosis in cholangiocarcinoma

doi: 10.1186/s10020-025-01362-8

Figure Lengend Snippet: Soluble AZGP1 facilitates the cell death of CCA cells. A Cell death was confirmed through trypan blue assay after 36 h of AZGP1-overexpressing in human CCA cell lines. B ELISA was measured on secretion levels of AZGP1 from AZGP1 OE in CCA cell lines. C Cell death quantification was performed after treatment with AZGP1-OE CM in KKU-213 and SNU-478 cell at varying concentrations for 72 h. D The presence of sAZGP1 in the AZGP1-OE CM was confirmed through ELISA analysis. E Immunoblot analysis assessing level of the indicated proteins in KKU-213 and SNU-478. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: To measure sAZGP1 in supernatant or whole blood samples, 96-well plated (SPL) were coated with a goat anti-AZGP1 polyclonal antibody (Lsbio) at 1 μg/ml.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Molecular Medicine

Article Title: Secreted AZGP1 induced by 5-FU binds to PD-L1 and promotes apoptosis in cholangiocarcinoma

doi: 10.1186/s10020-025-01362-8

Figure Lengend Snippet: In the CCA mouse model, the expression of sAZGP1 is significantly reduced. A The schematic illustrates the experimental design. A surgical procedure was performed to establish a mouse model of CCA in BALB/C mice ( n = 5 mice, per groups) B Representative images of depict the whole body of control and surgical mice (Left), the biliary duct (Top Right) and the liver (Bottom Right) at the study endpoint. C The quantification of sAZGP1 in the blood was conducted through ELISA. D The expression levels of AZGP1 and Ki-67 were assessed via western blot analysis in Liver and bile duct tissues. Tumors were collected 9 d post- surgery ( n = 5 mice per group). E Representative immunohistochemical (IHC) staining for AZGP1, CA19-9 (a CCA marker), and Ki-67 in bile duct tissues from control and surgical group mice (Left panel). Quantification of IHC staining was performed showing reduced AZGP1 and increased CA19-9 and Ki-67 expression in the surgical group (Right panel). Scale bar = 50 μm. ** P < 0.01, *** P < 0.001

Article Snippet: To measure sAZGP1 in supernatant or whole blood samples, 96-well plated (SPL) were coated with a goat anti-AZGP1 polyclonal antibody (Lsbio) at 1 μg/ml.

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay, Western Blot, Immunohistochemical staining, Immunohistochemistry, Marker

Journal: Molecular Medicine

Article Title: Secreted AZGP1 induced by 5-FU binds to PD-L1 and promotes apoptosis in cholangiocarcinoma

doi: 10.1186/s10020-025-01362-8

Figure Lengend Snippet: The upregulation of Foxo1 influences the transcription and secretion of AZGP1. A Cell death was quantified in CCA cells treated with 5-FU at a concentration of 10 µM for 72 h. B ELISA was conducted to measure the expression of sAZGP1 in CCA cells treated to 5-FU at 10 µM for 72 h. C Western blot analysis was performed on CCA cells following treatment with 5-FU at 10 µM for 72 h. D Western blot analysis of SNU-478 cells revealed time-dependent changes in the expression of p-Foxo1 and p-AKT after treatment with 5-FU. E Immunofluorescence was utilized to assess the localization of Foxo1 in SNU-478 cells, comparing non-treated controls (Con) with those treated with 5-FU at 10 µM for 24 h (5-FU). (Scale bar: 50 μm). Additionally, the mRNA F and protein levels G of AZGP1 were confirmed in relation to the overexpression of Foxo1. * P < 0.05, ** P < 0.01

Article Snippet: To measure sAZGP1 in supernatant or whole blood samples, 96-well plated (SPL) were coated with a goat anti-AZGP1 polyclonal antibody (Lsbio) at 1 μg/ml.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Immunofluorescence, Over Expression

Journal: Molecular Medicine

Article Title: Secreted AZGP1 induced by 5-FU binds to PD-L1 and promotes apoptosis in cholangiocarcinoma

doi: 10.1186/s10020-025-01362-8

Figure Lengend Snippet: PD-L1 functions as a significant interaction partner of sAZGP1. A The expression of levels AZGP1 and PD-L1 in SNU-478 cells overexpressing AZGP1 for 36 h, as determined by western blot analysis. B Representative quantification of flow cytometry measuring PD-L1 surface expression in both control and AZGP1 overexpressing cells. C Co-immunoprecipitation results demonstrated the interaction between sAZGP1 and PD-L1 in SNU-478 cells 36 h following transfection. D A schematic of the functional domains of human AZGP1 and PD-L1. E-F Co-immunoprecipitation further indicated the interaction between AZGP1 △SP D1-HA and PD-L1 D1-Myc in 293 T cells, 24 h post-transfection G Illustrating the action of sAZGP1 secreted from CCA cell lines interacting with membrane PD-L1. ** P < 0.01

Article Snippet: To measure sAZGP1 in supernatant or whole blood samples, 96-well plated (SPL) were coated with a goat anti-AZGP1 polyclonal antibody (Lsbio) at 1 μg/ml.

Techniques: Expressing, Western Blot, Flow Cytometry, Control, Immunoprecipitation, Transfection, Functional Assay, Membrane

Journal: Molecular Medicine

Article Title: Secreted AZGP1 induced by 5-FU binds to PD-L1 and promotes apoptosis in cholangiocarcinoma

doi: 10.1186/s10020-025-01362-8

Figure Lengend Snippet: The inhibition of sAZGP1 and PD-L1 results in the attenuation of cell death in CCA cells. A After shAZGP1 transfection for 24 h in KKU-213 and SNU-478 cell lines, the expression level of sAZGP1 was confirmed after treatment with 10 µM of 5-FU. B Reduced cell death was confirmed following shAZGP1 and 5-FU treatment. C The reduced expression of AZGP1 and PD-L1 following shAZGP1 and 5-FU treatment was confirmed by western blot. D The reduced expression of membrane PD-L1 following shAZGP1 and 5-FU treatment was confirmed by FACS. E Changes in p-AKT and cleaved caspase 3 were confirmed by western blot after shPD-L1 transfection for 24 h followed by 5-FU CM treatment. F Cell death was confirmed following shPD-L1 transfection and 5-FU CM treatment. ** P < 0.01, *** P < 0.001

Article Snippet: To measure sAZGP1 in supernatant or whole blood samples, 96-well plated (SPL) were coated with a goat anti-AZGP1 polyclonal antibody (Lsbio) at 1 μg/ml.

Techniques: Inhibition, Transfection, Expressing, Western Blot, Membrane

Journal: Molecular Medicine

Article Title: Secreted AZGP1 induced by 5-FU binds to PD-L1 and promotes apoptosis in cholangiocarcinoma

doi: 10.1186/s10020-025-01362-8

Figure Lengend Snippet: sAZGP1 influences T-cell activation within the microenvironment of CCA. A The expression of membrane PD-L1 in CCA cell lines was confirmed through FACS. B Cell death was quantified following treatment with recombinant AZGP1 in cell lines. C Changes in p-AKT following recombinant AZGP1 treatment were validated using western blot analysis. D A schematic representation of the experimental design was provided to confirm the activation of immune cells in peripheral blood mononuclear cells (PBMC) following recombinant AZGP1 treatment. E FACS of activated immune cells expressing CD45 + CD4 + and CD45 + CD8 + markers in PMBC following treatment with recombinant AZGP1. F A schematic depiction illustrates that the upregulation of sAZGP1 enhances the synergistic anticancer effects of PD-L1-mediated cell death and the activation of CD4 + T cells. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: To measure sAZGP1 in supernatant or whole blood samples, 96-well plated (SPL) were coated with a goat anti-AZGP1 polyclonal antibody (Lsbio) at 1 μg/ml.

Techniques: Activation Assay, Expressing, Membrane, Recombinant, Western Blot

Journal: Investigative Ophthalmology & Visual Science

Article Title: AZGP1 Attenuates Subretinal Fibrosis and Inhibits Epithelial-Mesenchymal Transition by Blocking the PI3K/AKT Signaling Pathway

doi: 10.1167/iovs.66.4.83

Figure Lengend Snippet: Sequence of Primers Used in the Experiments

Article Snippet: The cryosections were blocked with 3% BSA for 30 minutes at room temperature and then incubated with primary mouse antibody to AZGP1 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit antibody to RPE65 (1:200; Proteintech, Chicago, IL, USA) overnight at 4°C, followed by Alexa Fluor 488 anti-mouse secondary antibody (1:500; Beyotime, Shanghai, China) and Alexa Fluor 594 anti-rabbit secondary antibody (1:500; Abcam, CA, USA).

Techniques: Sequencing

Journal: Investigative Ophthalmology & Visual Science

Article Title: AZGP1 Attenuates Subretinal Fibrosis and Inhibits Epithelial-Mesenchymal Transition by Blocking the PI3K/AKT Signaling Pathway

doi: 10.1167/iovs.66.4.83

Figure Lengend Snippet: Sequence of Human siRNAs Used in the Experiments

Article Snippet: The cryosections were blocked with 3% BSA for 30 minutes at room temperature and then incubated with primary mouse antibody to AZGP1 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit antibody to RPE65 (1:200; Proteintech, Chicago, IL, USA) overnight at 4°C, followed by Alexa Fluor 488 anti-mouse secondary antibody (1:500; Beyotime, Shanghai, China) and Alexa Fluor 594 anti-rabbit secondary antibody (1:500; Abcam, CA, USA).

Techniques: Sequencing, Negative Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: AZGP1 Attenuates Subretinal Fibrosis and Inhibits Epithelial-Mesenchymal Transition by Blocking the PI3K/AKT Signaling Pathway

doi: 10.1167/iovs.66.4.83

Figure Lengend Snippet: AZGP1 was abnormally expressed in RPE/choroid from SRF mice compared with the healthy control s . Eyes were collected 3 days (SRF3D), 7 days (SRF7D), 14 days (SRF14D), 21 days (SRF21D), and 28 days (SRF28D) after the 2-stage laser induction. ( A – C ) Western blotting of the RPE/choroid complex showing discrepancies in AZGP1 expression after SRF induction ( N = 3 to 4). ( D ) Immunofluorescence staining showed expression of AZGP1 ( green ; white arrow ) and α-SMA ( red ) in cross-sections of SRF and the control mouse eyes. Cell nuclei were counterstained with DAPI ( blue ; N = 3). GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. Scale bar = 50 µm. All data are presented as mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001, the P value was obtained by 1-way ANOVA followed by multiple comparisons for multiple groups and two-sided t -test between individual groups.

Article Snippet: The cryosections were blocked with 3% BSA for 30 minutes at room temperature and then incubated with primary mouse antibody to AZGP1 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit antibody to RPE65 (1:200; Proteintech, Chicago, IL, USA) overnight at 4°C, followed by Alexa Fluor 488 anti-mouse secondary antibody (1:500; Beyotime, Shanghai, China) and Alexa Fluor 594 anti-rabbit secondary antibody (1:500; Abcam, CA, USA).

Techniques: Control, Western Blot, Expressing, Immunofluorescence, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: AZGP1 Attenuates Subretinal Fibrosis and Inhibits Epithelial-Mesenchymal Transition by Blocking the PI3K/AKT Signaling Pathway

doi: 10.1167/iovs.66.4.83

Figure Lengend Snippet: AZGP1 alleviated subretinal fibrosis in SRF mouse model. ( A ) Experimental protocols. Mice were subjected to the two-stage laser induction. The rAZGP1 (1 µg in 1 µL dilution buffer per eye), denatured rAZGP1 (de rAZGP1) or dilution buffer (vehicle) was intravitreally injected 1 day post-second laser. At day 14, choroid/RPE flat-mounts were made for further immunofluorescence staining, and RPE/choroid complex were collected for further Western blotting. ( B – D ) Representative immunostaining images and quantification for collagen I ( red ) and CD31 ( green ) in choroid/RPE flat-mounts of mice with and without intravitreal injection (IVI) of rAZGP1 after SRF induction. Cell nuclei were counterstained with DAPI ( blue ). Scale bar = 100 µm, N = 21 to 27 lesions from 8 eyes per group. ( E – G ) Western blotting of the RPE/choroid complex and quantification showing significant decrease of collagen I and α-SMA after IVI of rAZGP1 ( N = 3). All data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, the P value was obtained by 1-way ANOVA followed by multiple comparisons.

Article Snippet: The cryosections were blocked with 3% BSA for 30 minutes at room temperature and then incubated with primary mouse antibody to AZGP1 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit antibody to RPE65 (1:200; Proteintech, Chicago, IL, USA) overnight at 4°C, followed by Alexa Fluor 488 anti-mouse secondary antibody (1:500; Beyotime, Shanghai, China) and Alexa Fluor 594 anti-rabbit secondary antibody (1:500; Abcam, CA, USA).

Techniques: Injection, Immunofluorescence, Staining, Western Blot, Immunostaining

Journal: Investigative Ophthalmology & Visual Science

Article Title: AZGP1 Attenuates Subretinal Fibrosis and Inhibits Epithelial-Mesenchymal Transition by Blocking the PI3K/AKT Signaling Pathway

doi: 10.1167/iovs.66.4.83

Figure Lengend Snippet: AZGP1 was co-localized with RPE65 both in human and mouse eye samples. ( A ) The expression of Azgp1 was analyzed in several clusters associated with fibrosis of human RPE/choroid complex from GSE135922. ( B ) Co-localization of AZGP1 ( red dots ) and RPE65 ( green dots ) in RPE and choroidal cell populations from patients with neovascular AMD and controls (single-cell RNA-seq data from NCBI GEO database, ID: GSE135922). CTRL, normal macula, N = 5; nAMD, neovascular age-related macular degeneration, N = 2. ( C ) Immunofluorescence staining showed co-localization ( white arrow ) of AZGP1 ( green ) and RPE65 ( red ) in cross-sections of SRF mouse eyes (7 days after the second laser induction). ( D ) Immunofluorescence staining showed co-localization ( white arrow ) of α-SMA ( green ) and RPE65 ( red ) in SRF mouse eyes (7 days after the second laser induction). Cell nuclei were counterstained with DAPI ( blue ; N = 3). Scale bar = 50 µm.

Article Snippet: The cryosections were blocked with 3% BSA for 30 minutes at room temperature and then incubated with primary mouse antibody to AZGP1 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit antibody to RPE65 (1:200; Proteintech, Chicago, IL, USA) overnight at 4°C, followed by Alexa Fluor 488 anti-mouse secondary antibody (1:500; Beyotime, Shanghai, China) and Alexa Fluor 594 anti-rabbit secondary antibody (1:500; Abcam, CA, USA).

Techniques: Expressing, RNA Sequencing, Immunofluorescence, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: AZGP1 Attenuates Subretinal Fibrosis and Inhibits Epithelial-Mesenchymal Transition by Blocking the PI3K/AKT Signaling Pathway

doi: 10.1167/iovs.66.4.83

Figure Lengend Snippet: AZGP1 inhibited TGFβ1 induced EMT in RPE cells. Cells were treated with 10 ng/mL TGFβ1 for 48 hours to induce EMT with or without 1 µg/mL rAZGP1. ( A ) The mRNA level of AZGP1 was detected by RT-qPCR in the healthy controls, the TGFβ1, and the TGFβ1 + rAZGP1 groups in ARPE-19 cells ( N = 3). ( B , C ) Western blotting showed protein level of AZGP1 in the healthy controls, the TGFβ1, and the TGFβ1 + rAZGP1 groups in ARPE-19 cells ( N = 3 to 4). ( D – G ) Immunofluorescence staining showed reduced expression of α-SMA ( green ) with rAZGP1 treatment in ARPE-19 cells ( D , E ) and primary mouse RPE cells ( F , G ). Scale bar = 50 µm, N = 3. ( H , I ) mRNA expression of Col1a1 ( H ) and Fn1 ( I ) were detected by RT-qPCR in ARPE-19 from the healthy control, the TGFβ1, and the TGFβ1 + rAZGP1 groups ( N = 3). ( J – M ) Western blotting showed the protein expression of Fibronectin1, collagen I, and α-SMA in ARPE-19 from the healthy control, the TGFβ1, and the TGFβ1 + rAZGP1 groups ( N = 4). Control, control group; TGFβ1, TGFβ1 induction; TGFβ1 + rAZGP1, TGFβ1 induction with rAZGP1 treatment. All data are presented as mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001, the P value was obtained by 1-way ANOVA followed by multiple comparisons.

Article Snippet: The cryosections were blocked with 3% BSA for 30 minutes at room temperature and then incubated with primary mouse antibody to AZGP1 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit antibody to RPE65 (1:200; Proteintech, Chicago, IL, USA) overnight at 4°C, followed by Alexa Fluor 488 anti-mouse secondary antibody (1:500; Beyotime, Shanghai, China) and Alexa Fluor 594 anti-rabbit secondary antibody (1:500; Abcam, CA, USA).

Techniques: Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Expressing, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: AZGP1 Attenuates Subretinal Fibrosis and Inhibits Epithelial-Mesenchymal Transition by Blocking the PI3K/AKT Signaling Pathway

doi: 10.1167/iovs.66.4.83

Figure Lengend Snippet: AZGP1 inhibited the PI3K-AKT pathway in ARPE-19 cells. ( A ) KEGG pathway analysis showing the top 20 significant pathways of 66 common DEGs in SRF10D and SRF30D compared with the healthy controls. ( B , C ) Western blotting showed the protein expression of phospho-AKT (Ser473) and AKT in ARPE-19 from the healthy control, the TGFβ1, and the TGFβ1 + rAZGP1 groups ( N = 4). Control, control group; TGFβ1, TGFβ1 (10 ng/mL) induction for 48 hours; TGFβ1 + rAZGP1, TGFβ1 (10 ng/mL) induction with rAZGP1 (1 µg/mL) treatment for 48 hours. ( D – G ) ARPE-19 cells were transfected with negative control siRNA (NC) and Azgp1 targeted siRNA. TGFβ1 (10 ng/ml) and Capivasertib (1 µM) were added after RNA interference for additional 48 hours. Western blotting showed the phosphorylation of AKT ( D, F ) and PI3K ( E, G ) in ARPE-19 cells ( N = 3). ( H , I ) Western blotting showed the expression level of Collagen I, phosphor-AKT, AKT, phosphor-PI3K, and PI3K in ARPE-19 cells transduced with LV-Azgp1 and LV-empty after treated with 10 ng/mL TGFβ1 for 48 hours ( N = 3). The result of p-AKT/AKT was based on the band of p-AKT and AKT (incubated after stripping), whereas the result of p-PI3K/PI3K was based on the band of p-PI3K and PI3K normalized to GAPDH, respectively. All data are presented as mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001, the P value was obtained by 1-way ANOVA followed by multiple comparisons for multiple groups and two-sided t -test between individual groups.

Article Snippet: The cryosections were blocked with 3% BSA for 30 minutes at room temperature and then incubated with primary mouse antibody to AZGP1 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit antibody to RPE65 (1:200; Proteintech, Chicago, IL, USA) overnight at 4°C, followed by Alexa Fluor 488 anti-mouse secondary antibody (1:500; Beyotime, Shanghai, China) and Alexa Fluor 594 anti-rabbit secondary antibody (1:500; Abcam, CA, USA).

Techniques: Western Blot, Expressing, Control, Transfection, Negative Control, Phospho-proteomics, Transduction, Incubation, Stripping Membranes