Journal: Redox biology

Article Title: Hydrogen sulfide attenuates disturbed flow-induced vascular remodeling by inhibiting LDHB-mediated autophagic flux.

doi: 10.1016/j.redox.2024.103456

Figure Lengend Snippet: Fig. 6. NaHS inhibited the interaction between LDHB and ATP6V1A and reduced ATP6V1A-dependent lysosomal acidification. A, B. MOVAs were treated with NaHS (200 μM) and PDGF (20 ng/mL) for 24 h. Whole-cell lysates were immunoprecipitated and then immunoblotted with antibodies against the indicated proteins. C. PLA was performed to detect the interaction of LDHB and ATP6V1A. Data were quantified in the lower panel. ***p < 0.001.Scale bar = 50 μm. D. Co-localization of LDHB and ATP6V1A in MOVAs was examined by immunofluorescence. Data were quantified in the lower panel. n = 6, ***p < 0.001. Scale bars = 50 μm. E. LDHB- GFP plasmid was transfected into MOVAs for 24 h and then stimulated with PDGF in the presence or absence of NaHS. Lyso-Tracker Red was used to label lysosomes. Data were quantified in the lower panel. n = 6, ***p < 0.001.Scale bars = 20 μm. F. Lysosomal pH of MOVAs was measured by FITC-dextran staining (0.5 mg/mL). Data were quantified in the right panel. n = 6, ***p < 0.001. Scale bars = 50 μm.

Article Snippet: Primary antibodies were: LDHB (Proteintech, 14824) and ATP6V1A (Santa Cruz, sc293336).

Techniques: Immunoprecipitation, Immunofluorescence, Plasmid Preparation, Transfection, Staining

Journal: Redox biology

Article Title: Hydrogen sulfide attenuates disturbed flow-induced vascular remodeling by inhibiting LDHB-mediated autophagic flux.

doi: 10.1016/j.redox.2024.103456

Figure Lengend Snippet: Fig. 7. Identification of the domains of ATP6V1A and LDHB involved in their interaction. A. Schematic diagram illustrating the design of full-length HA-tagged ATP6V1A and the ATP6V1A truncates tagged with HA. B. HEK293T cells were co-transfected with these constructs, followed by IP assay using anti-Flag beads. Subsequent immunoblotting was performed with both anti-Flag and anti-HA antibodies to detect the expression of the constructs. C. The predicted structures of ATP6V1A and LDHB were generated using Alphafold. D. The conserved sequences containing L57 of ATP6V1A and S269 of LDHB were shown. E, F HEK293T cells were co-transfected with corresponding constructs and the cell lysates were collected for IP assays using beads conjugated with anti-Flag and anti-HA antibodies.

Article Snippet: Primary antibodies were: LDHB (Proteintech, 14824) and ATP6V1A (Santa Cruz, sc293336).

Techniques: Transfection, Construct, Western Blot, Expressing, Generated

Journal: Redox biology

Article Title: Hydrogen sulfide attenuates disturbed flow-induced vascular remodeling by inhibiting LDHB-mediated autophagic flux.

doi: 10.1016/j.redox.2024.103456

Figure Lengend Snippet: Fig. 8. The expression of LDHB was up-regulated in VSMCs of human TAA and AAA. A. Co-localization of LDHB and ACTA2 in human TAA and AAA were analyzed by immunofluorescence staining. Data were quantified in the right panel. n = 6, ***p < 0.001. Scale bars = 50 μm. B. The mRNA level of LDHB in aortic tissues was detected by RT-PCR. n = 5, ***p < 0.001. C. Schematic diagram of this study. H2S attenuates DF-induced vascular remodeling by inhibiting the interaction between LDHB and ATP6V1A. The disruption of this interaction prevents lysosomal acidification and autophagic flux, thereby suppressing VSMC proliferation and migration.

Article Snippet: Primary antibodies were: LDHB (Proteintech, 14824) and ATP6V1A (Santa Cruz, sc293336).

Techniques: Expressing, Immunofluorescence, Staining, Reverse Transcription Polymerase Chain Reaction, Disruption, Migration

Journal: Acta biomaterialia

Article Title: A subtype specific probe for targeted magnetic resonance imaging of M2 tumor-associated macrophages in brain tumors.

doi: 10.1016/j.actbio.2025.01.003

Figure Lengend Snippet: Fig. 6. Immunohistochemical and FACS validations of M2pep-uIONP targeting M2 TAMs. Confocal microscopic images of tumor tissues collected from mice at 24 h after i.v. injection of probes show (A) Cy7-M2pep-uIONP were highly co-localizing with CD68- and CD163-double positive M2 TAM, revealing the targeting specificity. In comparison, the non-targeted control probe Cy7-scM2pep-uIONP and Cy7-Ferumoxytol not only exhibited much less tumoral accumulation, but also were not co-localized with M2 TAM. Scale bars: 37 µm. (B) Flow cytometric zebra plots and (C) the corresponding quantification of CD68- and CD163-double positive M2 TAM targeted by FITC-M2pep-uIONP, FITC-scM2pep-uIONP, and FITC-Ferumoxytol. The gating strategy involves sorting CD163+ M2 TAM population in live cells dissociated from mouse cerebrums by gating on CD68+ pan-macrophage populations, followed by further gating to assess the FITC-labeled nanoparticles.

Article Snippet: Rabbit polyclonal anti-CD163 antibody was purchased from Boster Biological Technology (Pleasanton, CA, USA).

Techniques: Immunohistochemical staining, Injection, Comparison, Control, Labeling

Journal: Brain research bulletin

Article Title: The effects and mechanisms of AM1241 in alleviating cerebral ischemia-reperfusion injury.

doi: 10.1016/j.brainresbull.2024.111025

Figure Lengend Snippet: Fig. 4. Effects of AM1241 on the NF-κB and MAPK signaling pathways. (A): EMSA technique to examine NF-κB transcriptional activity. (B): Western blot analysis to detect the phosphorylation of MAPKs (ERK, JNK, P38). n=3, *P<0.05, **P<0.01, ***P<0.001. n=3.

Article Snippet: Primary antibodies against p-p38 (28796–1-AP, Proteintech), p38 (14064–1-AP, Proteintech), p-ERK (28733–1-AP, Proteintech), ERK (11257–1-AP, Proteintech), p-JNK (80024–1-RR, Proteintech), JNK (66210–1-Ig, Proteintech), IL-6 (ab233706, Abcam), TNF-α (ab183218, Abcam), NOX2 (ab310337, Abcam), BAX (50599–2- Ig, Proteintech), Cleaved-Caspase3 (C-Caspase3) (25128–1-AP, Proteintech) were added and incubated at 4◦C overnight.

Techniques: Protein-Protein interactions, Activity Assay, Western Blot, Phospho-proteomics

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: The ARPKD Protein DZIP1L Regulates Ciliary Protein Entry by Modulating the Architecture and Function of Ciliary Transition Fibers.

doi: 10.1002/advs.202308820

Figure Lengend Snippet: Figure 1. C. elegans DZIP-1 is an evolutionarily conserved basal body protein. A) The sequence alignment of two evolutionarily conserved domain (DZIP- like domain and the C2H2 zine finger domain) within C. elegans DZIP-1, H. sapiens DZIP1/DZIP1L and M. musculus DZIP1/DZIP1L. The Dzip-like_N domain (red line) is the most conserved domain in DZIP1/DZIP1L proteins. The ZnF_C2H2 domain is indicated by an orange line. And the highly conserved region is enclosed in the purple box. Asterisks are used to indicate the residues that, when mutated, can cause human ARPKD. B) DZIP-1 localizes to the ciliary base in C. elegans. The schematic diagram of phasmid cilia is depicted on the left, GFP-tagged DZIP-1 specifically localizes to

Article Snippet: Protein localization was confirmed by immunofluorescence imaging. gRNA sequence was as follows: DZIP1L gRNA#1, 5′- TGATCATCTTGCGACGCCGG-3′; DZIP1L gRNA#2, 5′- CAGTCCATGCTATCATGGCG-3′; ANKRD26 gRNA#1, 5′-GGGTAGCTCACAATCCTCTG-3′; ANKRD26 gRNA#2, 5′- AAATTCTTGTAACTAGAGTG-3′; Antibodies: The following primary antibodies were used in this study: ANKRD26 (rabbit, GeneTex, GTX128255, 1:500), DZIP1L (rabbit, Proteintech, 17474-1-AP, 1:500), FBF1 (rabbit, Proteintech, 11531-1-AP, 1:500), ARL13B (rabbit, Proteintech, 17711-1-AP, 1:500), KIAA0586/TALPID3 (rabbit, Proteintech, 24421-1-AP, 1:500), SCLT1 (rabbit, Proteintech, 14875- 1-AP, 1:500), CEP164 (rabbit, Proteintech, 22227-1-AP, 1:500), CEP89 (rabbit, Abcam, ab204410, 1:500), ODF2 (rabbit, Abcam, ab43840, 1:500), ODF2 (mouse, Abnova, H00004957-M01, 1:500), γ-tublin (mouse, Sigma-Aldrich, T6557, 1:1,000), FAM92A (rabbit, Proteintech, 24803-1- AP, 1:500), CBY1 (rabbit, Proteintech, 12239-1-AP, 1:500), GFP(mouse, Roche, 11814460001,1:200), IFT140 (rabbit, Proteintech, 17460-1-AP, 1:500), GT335(mouse, adipogen life science, A40251903), GLI3 (AF3690, R&D Systems), PC2 (Baltimore Polycystic Kidney Disease (PKD) Research and Clinical Core Center), Ac-tubulin (mouse, sigma, T7451, 1:300), Flag (mouse, sigma, F1804, 1:300), CEP290 (rabbit, abcam, ab84870).

Techniques: Sequencing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: The ARPKD Protein DZIP1L Regulates Ciliary Protein Entry by Modulating the Architecture and Function of Ciliary Transition Fibers.

doi: 10.1002/advs.202308820

Figure Lengend Snippet: Figure 6. The precise localization of DZIP1L in human RPE cells. Spatial localization of DZIP1L was examined using a combination of Ultrastructure expansion microscopy (ExM) and confocal microscopy. As our DZIP1L polyclonal antibody is derived from rabbits, and most of our antibodies for TF and TZ markers are also polyclonal antibodies from rabbit, co-labeling them with antibodies is not feasible. To assess their co-localization, we employed transgenic FLAG-DZIP1L and utilized a mouse monoclonal FLAG antibody (IgG1 isotype) to label DZIP1L. While cilia were marked using anti-Ac-tubulin, a mouse monoclonal antibody with IgG2b isotype. A) FLAG-DZIP1L was localized beneath the classical TZ component CEP290 in both ciliated and non-

Article Snippet: Protein localization was confirmed by immunofluorescence imaging. gRNA sequence was as follows: DZIP1L gRNA#1, 5′- TGATCATCTTGCGACGCCGG-3′; DZIP1L gRNA#2, 5′- CAGTCCATGCTATCATGGCG-3′; ANKRD26 gRNA#1, 5′-GGGTAGCTCACAATCCTCTG-3′; ANKRD26 gRNA#2, 5′- AAATTCTTGTAACTAGAGTG-3′; Antibodies: The following primary antibodies were used in this study: ANKRD26 (rabbit, GeneTex, GTX128255, 1:500), DZIP1L (rabbit, Proteintech, 17474-1-AP, 1:500), FBF1 (rabbit, Proteintech, 11531-1-AP, 1:500), ARL13B (rabbit, Proteintech, 17711-1-AP, 1:500), KIAA0586/TALPID3 (rabbit, Proteintech, 24421-1-AP, 1:500), SCLT1 (rabbit, Proteintech, 14875- 1-AP, 1:500), CEP164 (rabbit, Proteintech, 22227-1-AP, 1:500), CEP89 (rabbit, Abcam, ab204410, 1:500), ODF2 (rabbit, Abcam, ab43840, 1:500), ODF2 (mouse, Abnova, H00004957-M01, 1:500), γ-tublin (mouse, Sigma-Aldrich, T6557, 1:1,000), FAM92A (rabbit, Proteintech, 24803-1- AP, 1:500), CBY1 (rabbit, Proteintech, 12239-1-AP, 1:500), GFP(mouse, Roche, 11814460001,1:200), IFT140 (rabbit, Proteintech, 17460-1-AP, 1:500), GT335(mouse, adipogen life science, A40251903), GLI3 (AF3690, R&D Systems), PC2 (Baltimore Polycystic Kidney Disease (PKD) Research and Clinical Core Center), Ac-tubulin (mouse, sigma, T7451, 1:300), Flag (mouse, sigma, F1804, 1:300), CEP290 (rabbit, abcam, ab84870).

Techniques: Microscopy, Confocal Microscopy, Derivative Assay, Labeling, Transgenic Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: The ARPKD Protein DZIP1L Regulates Ciliary Protein Entry by Modulating the Architecture and Function of Ciliary Transition Fibers.

doi: 10.1002/advs.202308820

Figure Lengend Snippet: Figure 7. Conserved role of DZIP1L-ANKRD26 module in cilia gating in human RPE cells. A) The genetic interaction between DZIP1L and ANKRD26 in cilia formation is conserved in mammalian cells. The knockouts of either DZIP1L or ANKRD26 led to a moderate decrease in ciliation ratio. However, when both DZIP1L and ANKRD26 were knocked out, cilia formation was nearly completely blocked. The ciliation ratio was shown in the lower panel. Data are presented as mean ± SEM. n > 300 cells from three independent experiments. Significant differences were determined by two tailed t-test analysis. Scale bars: 5μm. B) Ciliary entry of the IFT component IFT140 was compromised in double mutants of DZIP1L and ANKRD26, compared to either of

Article Snippet: Protein localization was confirmed by immunofluorescence imaging. gRNA sequence was as follows: DZIP1L gRNA#1, 5′- TGATCATCTTGCGACGCCGG-3′; DZIP1L gRNA#2, 5′- CAGTCCATGCTATCATGGCG-3′; ANKRD26 gRNA#1, 5′-GGGTAGCTCACAATCCTCTG-3′; ANKRD26 gRNA#2, 5′- AAATTCTTGTAACTAGAGTG-3′; Antibodies: The following primary antibodies were used in this study: ANKRD26 (rabbit, GeneTex, GTX128255, 1:500), DZIP1L (rabbit, Proteintech, 17474-1-AP, 1:500), FBF1 (rabbit, Proteintech, 11531-1-AP, 1:500), ARL13B (rabbit, Proteintech, 17711-1-AP, 1:500), KIAA0586/TALPID3 (rabbit, Proteintech, 24421-1-AP, 1:500), SCLT1 (rabbit, Proteintech, 14875- 1-AP, 1:500), CEP164 (rabbit, Proteintech, 22227-1-AP, 1:500), CEP89 (rabbit, Abcam, ab204410, 1:500), ODF2 (rabbit, Abcam, ab43840, 1:500), ODF2 (mouse, Abnova, H00004957-M01, 1:500), γ-tublin (mouse, Sigma-Aldrich, T6557, 1:1,000), FAM92A (rabbit, Proteintech, 24803-1- AP, 1:500), CBY1 (rabbit, Proteintech, 12239-1-AP, 1:500), GFP(mouse, Roche, 11814460001,1:200), IFT140 (rabbit, Proteintech, 17460-1-AP, 1:500), GT335(mouse, adipogen life science, A40251903), GLI3 (AF3690, R&D Systems), PC2 (Baltimore Polycystic Kidney Disease (PKD) Research and Clinical Core Center), Ac-tubulin (mouse, sigma, T7451, 1:300), Flag (mouse, sigma, F1804, 1:300), CEP290 (rabbit, abcam, ab84870).

Techniques: Two Tailed Test