af5478  (R&D Systems)

Journal: Cell Communication and Signaling : CCS

Article Title: VEGFR3 mitigates hypertensive nephropathy by enhancing mitophagy via regulating crotonylation of HSPA1L

doi: 10.1186/s12964-025-02045-x

Figure Lengend Snippet: VEGFR3 interacts with HSPA1L to alleviate oxidative stress injury by regulating PARKIN-dependent mitophagy in vitro. A HEK-293T cells were transfected with plasmids encoding FLAG-VEGFR3 and HA-HSPA1L and prepared for immunoprecipitation and immunoblotting assays with the indicated antibodies. B-G HK-2 cells were co-transfected with FLAG-VEGFR3 or FLAG-Vector together with si-NC or si-HSPA1L, and then incubated with Ang II. B , C Colocalization of PARKIN (red) and TOMM20 (green) in HK-2 cells. Scale bar, 10 μm. D-G The levels of PARKIN, LC3 and SQSTM1 in mitochondrial lysates were examined by immunoblotting. The immunoblots were statistically quantified by densitometry. H-L HK-2 cells were transfected with or without plasmids encoding FLAG-VEGFR3 and si-HSPA1L, and then cells were incubated with Ang II, in the presence or absence of 3-MA. The immunoblots were statistically quantified by densitometry. Data were from three or more independent experiments and were presented as the mean ± SEM. Data in ( C - L ) were analyzed with one-way ANOVA. ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs. the indicated group

Article Snippet: ACTB (AF7018), GAPDH (AF7021), VDAC1 (AF5478), HSPA1L (DF6662), PARKIN (AF0235), PINK1 (DF7742) antibodies and Horseradish peroxidase (HRP)–conjugated immunoglobulin G (S0001) were acquired from Affinity Biosciences (OH.USA).

Techniques: In Vitro, Transfection, Immunoprecipitation, Western Blot, Plasmid Preparation, Incubation

Journal: Cell Communication and Signaling : CCS

Article Title: VEGFR3 mitigates hypertensive nephropathy by enhancing mitophagy via regulating crotonylation of HSPA1L

doi: 10.1186/s12964-025-02045-x

Figure Lengend Snippet: Crotonylation modification of HSPA1L at K130 by VEGFR3 is required for mitophagy regulation. A HEK-293T cells were transfected with FLAG-VEGFR3 and HA-HSPA1L and prepared for immunoprecipitation and immunoblotting. B The crotonylation modification at K130 was identified in HSPA1L by mass spectrometry. C The crotonylation of HSPA1L was assessed by immunoblotting with crotonylated lysine antibody following the HA immunomagnetic beads pulldown. in HK-2 cells expressing HSPA1L wild-type control (HA-WT), or the mutation (HA-K130R) with TSA (3 µM, 12 h) treatment. D , E HK-2 cells were transfected with plasmids encoding HSPA1L HA-WT or HA-K130R incubated with or without Ang II. Representative immunoblots of PARKIN, SQSTM1and LC3 II/LC3 I in mitochondria, as well as 3-nitrotyrosine and 8-oxo-dG in cell lysates. Immunoblots were statistically quantified by densitometry. F Alignment of sequences surrounding K130 in HSPA1L homologs from diverse species. Crotonylated lysine residue at HSPA1L-K130 are highlighted (red). G-H HK-2 cells were co-transfected with FLAG-VEGFR3 or FLAG-Vector together with HA-WT or HA-K130R, and then incubated with Ang II. Immunoblotting analysis and quantitative results of PARKIN, SQSTM1and LC3 II/LC3 I in mitochondria, and 3-nitrotyrosine in cell lysates. Data were from three independent experiments. Data in ( E ) and ( H ) were analyzed with one-way ANOVA. ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001 vs. the indicated group. I , J VEGFR3 contains three functional domains, including the Ig-like (IGI), Protein kinase (KD) and Disordered (DIS) domains. Full-length or truncated Flag-tagged VEGFR3 plasmid was transfected together with HA-HSPA1L. Total cell lysates were subjected to Co-IP with anti-FLAG antibody and immunoblotting was performed with FLAG or HA antibodies

Article Snippet: ACTB (AF7018), GAPDH (AF7021), VDAC1 (AF5478), HSPA1L (DF6662), PARKIN (AF0235), PINK1 (DF7742) antibodies and Horseradish peroxidase (HRP)–conjugated immunoglobulin G (S0001) were acquired from Affinity Biosciences (OH.USA).

Techniques: Modification, Transfection, Immunoprecipitation, Western Blot, Mass Spectrometry, Expressing, Control, Mutagenesis, Incubation, Residue, Plasmid Preparation, Functional Assay, Co-Immunoprecipitation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: VEGFR3 mitigates hypertensive nephropathy by enhancing mitophagy via regulating crotonylation of HSPA1L

doi: 10.1186/s12964-025-02045-x

Figure Lengend Snippet: The protective effect of VEGFR3 is attenuated in hypertensive mice with HSPA1L-K130R. AAV9 carried with HSPA1L-WT and HSPA1L-K130R were injected the renal pelvis of mice. 2 weeks after AAV9 transfection, these mice were infused with Ang II co-administered with VEGFC via osmotic minipumps. A Systolic BP of the indicated group mice were obtained by the tail-cuff method. B mRNA level of KIM-1 was determined in urine samples of mice. C , D Renal injury was evaluated by CRE and BUN in plasma. E , F HE staining and Masson staining were carried out to assess renal tubular injury, Scale bar, 2–200 μm. G , H The levels of PARKIN, SQSTM1and LC3 II/LC3 I in mitochondria of the renal cortex from each group mice were measured by Immunoblotting. I , J Immunoblotting analysis of 3-nitrotyrosine, 8-oxo-dG, and SOD1 protein expression levels in renal cortex and quantification. K , L Renal tissues were stained to detect MitoSOX Assay Kit (red). Scale bar, 100 μm. M Graphic abstract of the underlying mechanism. n = 5 mice per group. Data in ( A ) was performed by two-way ANOVA followed by Sidak multiple comparison test. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. the HSPA1L-WT group. Data in ( B-L ) were analyzed with Student’s t-test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs. the indicated group

Article Snippet: ACTB (AF7018), GAPDH (AF7021), VDAC1 (AF5478), HSPA1L (DF6662), PARKIN (AF0235), PINK1 (DF7742) antibodies and Horseradish peroxidase (HRP)–conjugated immunoglobulin G (S0001) were acquired from Affinity Biosciences (OH.USA).

Techniques: Injection, Transfection, Clinical Proteomics, Staining, Western Blot, Expressing, Mitosox Assay, Comparison

Journal: Cell Reports

Article Title: Quantitative Proteomics Analysis of Lytic KSHV Infection in Human Endothelial Cells Reveals Targets of Viral Immune Modulation

doi: 10.1016/j.celrep.2020.108249

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Article Snippet: Polyclonal sheep-anti-STX7 antibody , R and D Systems , Cat# AF5478; RRID: AB_2239977.

Techniques: Purification, Control, Virus, Recombinant, Mass Spectrometry, Sample Prep, Transfection, SYBR Green Assay, Sequencing, CRISPR, Plasmid Preparation, Software