migration tube formation western blot native page heat shock  (Lonza)

Journal: Cell Stress & Chaperones

Article Title: Dynamics of heat shock protein 70 kDa in heat-shocked and hypoxic human endothelial cells

doi: 10.1016/j.cstres.2025.100085

Figure Lengend Snippet: Temporal modulation of human HSP high-molecular-weight complexes by heat shock in primary ECs. (a) Invitrogen’s NativeMark™ was subjected to Native PAGE, stained with Coomassie blue, and used as a standard to identify high-molecular-weight complexes in native PAGE. (b) Whole-cell lysates from HUVECs exposed to heat shock, recovery, or basal conditions were subjected to native PAGE and sequentially stained for HSP40 (CST, 4868S), HSC70 (Invitrogen PA5-27337), HSP70 (Invitrogen MA3-009), and HSP90 (CST, 4877S). Below, sodium dodecyl sulfate-PAGE for each protein is displayed. The data are representative of n = 3–6 independent experiments with cells from different lots (19TL127368, 19TL028325, 19TL023264, and 21TL195720). (c) Merged image of all the stained samples shown in (b). The data are representative of n = 3–6 independent experiments in cells from different lots (19TL127368, 19TL028325, 19TL023264, and 21TL195720). (d) Quantification of the high-molecular-weight complex intensities from specific proteins from (b) versus Basal n. The data are presented as one-way ANOVA, with Tukey’s post hoc test; ** P < 0.01, *** P < 0.001 versus the respective control; HSP40, n = 3; HSP70, n = 4; HSC70, n = 5; and HSP90, n = 6. (E) Schematic representation of the native PAGE procedure. Proteins in their native state from cell lysates were separated on a 6% polyacrylamide gel and transferred to a nitrocellulose membrane overnight. The membrane was blocked with nonfat milk, incubated with primary antibody overnight, and then incubated with secondary antibody. The membrane was scanned using the LI-COR Odyssey® 2-channel near-infrared fluorescence imaging system, and the fluorescence levels of the last HSP probed were quantified. The same membrane was then incubated overnight with a primary antibody against another chaperone, followed by secondary antibody incubation, scanning with the near-infrared fluorescence imager, and quantification. This process was repeated daily until all chaperones were stained on the same membrane, in this order: day 1 anti-HSP40; day 2 anti-HSC70; day 3 anti-HSP90; day 4 anti-HSP70. Abbreviations used: EC, endothelial cell; HSP, heat-shock protein; HUVEC, human vein.

Article Snippet: HUVEC , Lonza , 21TL195720 , Migration Tube formation Western blot Native PAGE Heat shock.

Techniques: High Molecular Weight, Clear Native PAGE, Staining, Control, Membrane, Incubation, Fluorescence, Imaging

Journal: Cell Stress & Chaperones

Article Title: Dynamics of heat shock protein 70 kDa in heat-shocked and hypoxic human endothelial cells

doi: 10.1016/j.cstres.2025.100085

Figure Lengend Snippet: Hypoxia induces decreases in multimeric HSPs in human coronary arteries (HCAECs) and vein endothelial cells (HUVECs). (a) Whole-cell lysates from HCAECs exposed to hypoxia, heat-shock recovery, or basal conditions were subjected to native PAGE and sequentially stained for HSC70 (Invitrogen PA5-27337), HSP70 (Invitrogen MA3-009), HSP90 (CST 4877S), and HSP40 (CST 4868S), as shown in the top panels. Below, sodium dodecyl sulfate-PAGE results for each protein are displayed. Data are representative of n = 3 independent experiments using two different lots of cells (20TL365545 and 20TL064651, Lonza). Both native PAGE and Western blot experiments were performed using two different lots of cells (20TL365545 and 20TL064651). (b) Quantification of HSC70, HSP70, and HSP90 high-molecular-weight complexes in (a). Data are presented as the means ± SEMs, one-way ANOVA, with Tukey’s post hoc n = 3, * P < 0.05, ** P < 0.01 versus basal conditions. Unpaired t test ( # P < 0.05) was used to compare only the HSP90 hypoxic and basal groups. (c) Whole-cell lysates from HUVECs exposed to hypoxia or basal conditions were subjected to native PAGE and sequentially stained for HSC70 (Invitrogen PA5-27337) and HSP90 (CST 4877S), as shown in the top panels. Below, sodium dodecyl sulfate-PAGE for each protein is displayed. The data are representative of n = 2 independent experiments using cells from lot 19TL028324. (d) Whole-cell lysates of HUVECs exposed to hypoxia or basal conditions were subjected to Western blot analysis for HSP40 (CST 4868S), total HSP70 (Invitrogen MA3-006), HSC70 (Invitrogen PA5-27337), and HSP90 (CST 4877S). The data are representative of n = 3 independent experiments using cells from lots 19TL028324 and 23TL086130. (e) Data from (d) are presented as the mean ± SEM, unpaired Student’s t test, n = 3 with * P < 0.05 and *** P < 0.001. β-actin, loading control. (f) HUVECs subjected to basal or hypoxic 1% O 2 (94% N 2 , 5% CO 2 ) for 24 h were subjected to Western blot analysis for HIF1α (CST, #48085) with β-actin (Sigma, A5441) loading control. The assay was performed three times using two different lots of cells (21TL195720 and 23TL086130).

Article Snippet: HUVEC , Lonza , 21TL195720 , Migration Tube formation Western blot Native PAGE Heat shock.

Techniques: Clear Native PAGE, Staining, Western Blot, High Molecular Weight, Control