Journal: Journal of Lipid Research

Article Title: TNF-α induces acyl-CoA synthetase 3 to promote lipid droplet formation in human endothelial cells [S]

doi: 10.1194/jlr.RA119000256

Figure Lengend Snippet: TNF-α stimulation results in increased ACSL3 expression in HCAECs. A: HCAECs from donor 1 were stimulated for 18 h with 20 ng/ml TNF-α. ACSL and CCL2 mRNA levels were evaluated by real-time PCR. A representative experiment among three independent experiments is shown. B, C: ACSL3 and CCL2 mRNA levels were measured by real-time PCR at different time-points after 20 ng/ml of TNF-α stimulation, and expressed as fold over control cells at time-point 0. A representative experiment among two independent experiments is shown. The results are expressed as mean ± SEM (n = 3). HCAECs were transfected with ACSL3 siRNA using an Amaxa Nucleofector kit. RNA, protein, and supernatant samples were harvested 72 h after electroporation. Before the harvest, cells were stimulated with TNF-α (20 ng/ml) for 24 h. D: ACSL3 protein levels were measured by Western blot analysis and normalized to β-actin levels. Two representative blots among five independent experiments are shown. Molecular mass (kilodaltons) markers are indicated on the right. The lower molecular mass band on the second β-actin blot is a nonspecific band. E: Levels of ACSL3 mRNA were measured by real-time PCR and presented as fold over control. A representative experiment among three independent experiments is shown. Unpaired two-tailed Student’s t-test (A) and two-way ANOVA followed by Tukey’s multiple comparison test (B–E) were applied. *P < 0.05; **P < 0.01; ***P < 0.001 compared to vehicle-treated cells or time-point 0. ##P < 0.01 compared to control siRNA-treated cells. NS, no significant difference.

Article Snippet: Knockdown of ACSL3 by siRNA HCAEC cultures (80–90% confluence) were trypsinized and resuspended (5 × 10 3 cells/μl) in Amaxa HCAEC Nucleofector solution (Lonza, Cologne, Germany) containing 300 nM siRNA.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Electroporation, Western Blot, Two Tailed Test

Journal: STAR Protocols

Article Title: A metabolic labeling protocol to enrich myristoylated proteins from Caenorhabditis elegans

doi: 10.1016/j.xpro.2021.101013

Figure Lengend Snippet:

Article Snippet: Click-&-Go TM Click Chemistry Reaction Buffer Kit (Cat# 1001, Click Chemistry Tools) was used to detect proteins modified by myristate alkyne through western blot.

Techniques: Recombinant, Virus, Protein Enrichment, Staining, Software