anti ilk  (Santa Cruz Biotechnology)

Journal: International Journal of Molecular Sciences

Article Title: ILK Deletion Protects Against Chronic Kidney Disease-Associated Vascular Damage

doi: 10.3390/ijms27010215

Figure Lengend Snippet: ILK deletion prevented the development of vascular fibrosis in adenine-fed mice. ( A ) Wild-type (WT) and conditional ILK deletion (cKD-ILK) mice were fed a standard (Control) or adenine-rich (Adenine) diet for 6 weeks. Cross sections of aortas were analyzed histologically. Representative microphotographs of Sirius red (20×) staining are shown. Scale bar, 100 μm. Fibrosis in the tunica media of the aorta was determined by the intensity of collagen staining by Sirius red. Results were expressed as a percentage of the intensity of WT Control mice. Results are shown as mean ± SEM. n = 5–8 animals/group. * p < 0.05 vs. WT Control; # p < 0.05 vs. WT Adenine. ( B – D ) Wild-type (WT, solid lines, white symbols) and conditional ILK deletion (cKD-ILK, dashed lines, black symbols) mice were fed a standard (Control, circles) or adenine-rich (Adenine, triangles) diet for 2, 4 or 6 weeks. Collagen I (COL I) ( B ), fibronectin (FN) ( C ) and TGF-β1 ( D ) mRNA expression was measured in aortas, normalized against β-actin as endogenous control. Results are shown as mean ± SEM. n = 3–7 animals/group. * p < 0.05 vs. WT Control at the same time; $ p < 0.05 vs. 2 weeks; # p < 0.05 vs. WT Adenine at the same time.

Article Snippet: Primary antibodies used were against AKT, P-AKT (Ser473), COL I, GSK-3β, P-GSK-3β (Ser9), ILK (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Control, Staining, Expressing

Journal: International Journal of Molecular Sciences

Article Title: ILK Deletion Protects Against Chronic Kidney Disease-Associated Vascular Damage

doi: 10.3390/ijms27010215

Figure Lengend Snippet: ILK expression correlates with the expression of extracellular matrix proteins, collagen I (COL I) and fibronectin (FN), and TGF-β1 in the aortas. Wild-type and conditional ILK deletion mice were fed a standard or adenine-rich diet for 2, 4 or 6 weeks. ILK mRNA expression was confronted to the values of COL I ( A ), FN ( B ), and TGF-β1 ( C ) mRNA expressions in the aortas. The analysis is detailed in the Materials and methods section. n = 3–7 animals/group.

Article Snippet: Primary antibodies used were against AKT, P-AKT (Ser473), COL I, GSK-3β, P-GSK-3β (Ser9), ILK (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: ILK Deletion Protects Against Chronic Kidney Disease-Associated Vascular Damage

doi: 10.3390/ijms27010215

Figure Lengend Snippet: ILK deletion prevented fibrosis marker expression increase in mice aortas treated ex vivo with uremic toxins. Wild-type (WT) and ILK conditional-knockdown (cKD-ILK) mice were fed a standard diet. Aortas were excised and treated ex vivo with a combination of p -cresyl sulfate (pCS) and indoxyl sulfate (IS) at high doses (226 µg/mL and 100 µg/mL, respectively) for 24 h. After incubation with the uremic toxins, ILK ( A ), collagen I (COL I) ( B ), fibronectin (FN) ( C ), and TGF-β1 ( D ) mRNA expression in the aortas, normalized against β-actin as an endogenous control, was measured. Results are shown as mean ± SEM. * p < 0.05 vs. WT Control; & p < 0.05 vs. cKD-ILK Control; # p < 0.05 vs. WT (pCS+IS). n = 3–7 animals/group.

Article Snippet: Primary antibodies used were against AKT, P-AKT (Ser473), COL I, GSK-3β, P-GSK-3β (Ser9), ILK (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Marker, Expressing, Ex Vivo, Knockdown, Incubation, Control

Journal: International Journal of Molecular Sciences

Article Title: ILK Deletion Protects Against Chronic Kidney Disease-Associated Vascular Damage

doi: 10.3390/ijms27010215

Figure Lengend Snippet: p -cresyl sulfate (pCS) and indoxyl sulfate (IS) increase ILK activity and do not regulate its expression in HA-VSMC cells. HA-VSMC cells were incubated with a combination of pCS and IS at high doses (226 µg/mL and 100 µg/mL, respectively) for different times. After incubation with the uremic toxins, ILK expression was measured by Western blot ( A ) and RT-qPCR ( B ) and phosphorylation levels of GSK-3β at serine 9 (P-GSK-3β) ( C ) and AKT at serine 473 (P-AKT) ( D ) were measured by Western blot. ( A , C , D ) Representative Western blots of ILK, P-GSK-3β, and P-AKT are shown. GAPDH, GSK-3β, and AKT were used as the endogenous controls, respectively. The bars represent the normalized densitometric values of the blots against the endogenous control values. ( B ) ILK mRNA expression was quantified by RT-qPCR. Relative fold changes in mRNA content vs. untreated control (CT) are represented after the normalization with total β-actin content as the endogenous control. All values are presented as the mean ± SEM of 4 independent experiments. * p < 0.05 vs. CT.

Article Snippet: Primary antibodies used were against AKT, P-AKT (Ser473), COL I, GSK-3β, P-GSK-3β (Ser9), ILK (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Activity Assay, Expressing, Incubation, Western Blot, Quantitative RT-PCR, Phospho-proteomics, Control

Journal: International Journal of Molecular Sciences

Article Title: ILK Deletion Protects Against Chronic Kidney Disease-Associated Vascular Damage

doi: 10.3390/ijms27010215

Figure Lengend Snippet: p -cresyl sulfate (pCS) and indoxyl sulfate (IS) induce the expression of collagen I (COL I) through ILK in HA-VSMC cells. HA-VSMC cells were transfected with scrambled RNA (Sc, black bars) or were depleted of ILK with specific siRNA (ILK siRNA, white bars) and were incubated with a combination of pCS and IS at high doses (226 µg/mL and 100 µg/mL, respectively) for 48 h. ( A ) After incubation with the uremic toxins, protein expression of COL I was measured by Western blot. Representative Western blots of COL I are shown. GAPDH was used as the endogenous control. The bars represent the normalized densitometric values of the blots against the endogenous control values. ( B ) Prior to transfection, cells were seeded on coverslips and, after incubation with the uremic toxins, cells were labeled with an anti-COL I antibody (red) and nuclei with Hoechst 33342 (blue). Fluorescence intensity was determined by fluorescence microscopy. Images from a representative experiment are shown. Scale bar: 100 μm. The bar graph represents the average percentages of COL I fluorescence intensity normalized with respect to the number of cells in each image. Results were expressed as a percentage with respect to the Sc untreated control (CT). All values are presented as the mean ± SEM of 3 or 4 independent experiments. * p < 0.05 vs. Sc CT.

Article Snippet: Primary antibodies used were against AKT, P-AKT (Ser473), COL I, GSK-3β, P-GSK-3β (Ser9), ILK (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Transfection, Incubation, Western Blot, Control, Labeling, Fluorescence, Microscopy