Journal: Cell reports

Article Title: Blood flow patterns switch VEGFR2 activity through differential S-nitrosylation and S-oxidation

doi: 10.1016/j.celrep.2023.113361

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cell lysates were immunoprecipitated with 30 μL of streptavidin agarose beads (Millipore) at 4 ◦ C overnight.

Techniques: Virus, Recombinant, Biotin Switch Assay, Mutagenesis, Control, Sequencing, Software

Journal: Oncotarget

Article Title: The palmitoylation of the N-terminal extracellular Cys37 mediates the nuclear translocation of VPAC1 contributing to its anti-apoptotic activity

doi: 10.18632/oncotarget.17449

Figure Lengend Snippet: ( A ) After the biotinylated proteins from VPAC1-CHO and VPAC1-C37/A-CHO subjected to the acyl-biotin exchange assay with (HA+) or without (HA-) hydroxylamine treatment were pulled down by streptavidin-agarose and electrophoresed by SDS-PAGE (left) and then detected using anti-EYFP antibody (right), VPAC1-EYFP was significantly detectable, while VPAC1-C37/A-EYFP is almost undetectable, indicating that Cys37 was palmitoylated. And the negative interference of HA showed that the acyl-biotin exchange assay was HA depended and the palmitoylation of Cys37 is via hydroxylamine-sensitive thioester bond. ( B ) VPAC1-CHO cells were incubated for 12 hours with (Odya+) or without (Odya-) palmitate ortholog. Samples were divided and either not biotinylated through the click reaction (No RXN), reduced prior to the reaction with HA, or biotinylated and not reduced (RXN). Only samples treated with RXN without HA showed significant signal, supporting the specificity of the click chemistry palmitolaytion assay. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. ( C ) After VPAC1-CHO cells were incubated with Odya for 30, 60, 120 and 240 min hours, click reaction was performed followed by biotin pull-down and western blot analysis for EYFP. The time-dependent signals confirmed the technique's validity. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. ( D ) After VPAC1-CHO cells and VPAC1-C37/A-CHO cells were incubated with (Odya+) or without (Odya-) palmitate ortholog, click reaction was performed followed by biotin pull-down and western blot analysis for EYFP. VPAC1-EYFP was significant detectable, while VPAC1-C37/A-EYFP was almost undetectable, indicating the palmitoylation of Cys37 was determined by the click chemistry palmitolaytion assay. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. Representative blots from at least three independent experiments are shown.

Article Snippet: Then streptavidin-agarose mini-column (Sigma, USA) were used to gather the biotin-labeling proteins, and the final elution from the streptavidin-agarose mini-column was submitted to 10% SDS-PAGE and western blotting using monoclonal antibody recognizing EYFP (Amyjet Scientific, China).

Techniques: SDS Page, Incubation, Western Blot