Fig 1: Integrins and Talin1 contribute to the homing of CD4 T cells into the LN after i.l. injection.Quantitative analysis of adoptively transferred cells in popliteal LNs 90 min after i.l. injection of combinations of activated CD4+ T as indicated: a wild type and 4Itg-/-; b wild type and Talin-1-/-. a, b Left, total cell counts; dots represent cell counts per LN section; numbers above indicate the percentage of change compared to wild type (parench parenchyma, sin. sys. sinus system). c Migration distance of adoptively transferred cells from the SCS as indicated; dots represent cells; red bars, median; ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001 (paired t-test). Analysis of intranodal CD4+ T cell migration (left) and representative movies (right) (d–f) based on ex vivo time-lapse imaging of popliteal LNs after i.l. injection of activated wild type and Talin1-/- (d); wild type and 4Itg-/- (e), or PTX-treated wild type and 4Itg-/- (f) CD4+ T cells. Data calculated based on all cell tracks present within selected regions using automated tracking with manual correction. Dots represent the mean of one cell population per movie. ns not significant; a, b Wilcoxon signed rank test; c Mann Whitney test *p < 0.05; **p < 0.01, and ***p < 0.001 (paired t-test). Data are derived from three experiments with a total of eight (a, c) or four (d–f) mice, or from two experiments with a total of six mice (b, c). Scale bars 50 µm.
Fig 2: PSMC5 mediates Tln1 ubiquitination and degradation.(A) HUVEC lysates were incubated with in vitro-synthesized biotin-labelled sense or antisense AZIN2-sv for biotin pulldown followed by immunoblot analysis. (B) RIP assays were performed using an antibody against PSMC5 or negative control IgG. The purified RNA was used for qRT-PCR analysis, and the enrichment of AZIN2-sv was normalized against the input. ?p < .05 vs. RIP with IgG; n = 6 per group. (C) Agarose gel electrophoresis of RIP products. Arrow indicates the enriched AZIN2-sv. (D) PSMC5 protein levels detected by western blotting when AZIN2-sv was overexpressed and knocked down (GAPDH is the internal reference). (E) HUVECs with AZIN2-sv overexpression or knockdown were treated with MG132 (5 µM) for 24 h. Cell lysates were immunoprecipitated with an antibody against Tln1 or PSMC5. The precipitates and input were analysed by immunoblotting. (F) Immunoblots for PSMC5 and Tln1 in HUVECs transfected with PSMC5-targeting siRNAs or scrambled control siRNAs (GAPDH is the internal reference). ?p < .05 vs. control; n = 6 per group. (G) HUVECs transfected with PSMC5-targeting siRNAs or scrambled control siRNAs were treated with MG132 (5 µM) for 24 h. Cells lysates were immunoprecipitated with either control IgG or antibody against Tln1. The precipitates and input were analysed by immunoblotting.
Fig 3: A schematic representing the role of AZIN2-sv in angiogenesis.AZIN2-sv is an anti-angiogenesis lncRNA that acts by regulating Tln1 degradation through ubiquitination and binding to miR-214, resulting in the suppression of ITGB1 and inhibition of the Akt pathway and poor prognosis after MI. The transcription factor Bach1, a negative regulator of angiogenesis, can bind to the AZIN2-sv promoter and enhance its expression.
Fig 4: Tln1 is a downstream interactor protein of AZIN2-sv.(A) Silver-stained SDS-PAGE gel image representing proteins immunoprecipitated by AZIN2-sv and its antisense RNA in endothelial cells. The arrow indicates the region of the gel excised for western blotting. (B) Tln1 protein was detected in the specific band by western blotting. (C) RIP assays were performed using an antibody against Tln1 or negative control IgG. The purified RNA was used for qRT-PCR analysis, and enrichment of the AZIN2-sv was normalized against the input. ?p < .05 vs. RIP with IgG; n = 6 per group. (D) Agarose gel electrophoresis of RIP products. Arrow indicates enriched AZIN2-sv. (E) FISH and immunofluorescence for AZIN2-sv and Tln1 forty-eight hours after transfection with AZIN2-sv or control vector (bars, 40 µm). Blue indicates the nucleus, and red and green indicate the location of AZIN2-sv and Tln1, respectively. (F) ITGB1 and Tln1 protein levels detected by western blotting when AZIN2-sv was knocked down or overexpressed (GAPDH is the internal reference). (G) Quantification of protein levels in (F). ?p < .05 vs. vector. #p < .05 vs. shNC; n = 6 per group.
Fig 5: AZIN2-sv promotes Tln1 degradation through the ubiquitin-proteasome pathway.(A) Tln1 mRNA level was detected by qRT-PCR when AZIN2-sv was knocked down or overexpressed. (B) AZIN2-sv was overexpressed and silenced in HUVECs, and the cells were then treated with cycloheximide (CHX; 20 µg/mL) for the indicated time. Tln1 protein levels were analysed by immunoblotting (GAPDH is the internal reference). (C) Quantification of the protein level in (B). ?p < .05 vs. control; n = 6 per group. (D) AZIN2-sv or control vector was transfected into HUVECs, and the cells were then treated with MG132 (5 µM) or vehicle for 24 h. Cell lysates were analysed by immunoblotting. ?p < .05 vs. vector; n = 6 per group. (E, F) HUVECs with AZIN2-sv overexpression (E) or knockdown (F) were treated with MG132 (5 µM) for 24 h. Cell lysates were immunoprecipitated with either control IgG or an antibody against Tln1 and analysed by immunoblotting with a ubiquitin (Ub)-specific antibody. Bottom, input from cell lysates.
Supplier Page from Abcam for Anti-Talin 1 antibody [8D4]