Alkyne palmitoyl coa

Recombinant 6xHis-TEAD protein was treated with compounds under indicated concentrations in 50 mM MES buffer (pH 6.4) for 30 min. After incubation with 1 μM of alkyne palmitoyl-CoA (15968, Cayman) for 1 hr, 50 μL of sample mixture was treated with 5 μL of freshly prepared ‘click’ mixture containing 100 uM TBTA (678937, Sigma-Aldrich), 1 mM TCEP (C4706, Sigma-Aldrich), 1 mM CuSO₄ (496130, Sigma-Aldrich), 100 uM Biotin-Azide (1167-5, Click Chemistry Tools), and incubated for another 1 hr. The samples were then added 11 μL of 6xSDS loading buffer (BP-111R, Boston BioProducts) and denatured at 95°C for 5 min. SDS-PAGE was used to analyze the samples. Palmitoylation signal was detected by streptavidin-HRP antibody (1:3000, S911, Invitrogen). The total protein level was detected by primary anti-His-tag antibody (1:10,000, MA1-21315, Invitrogen) and secondary anti-mouse antibodies (1:5000, 7076S, Cell Signaling). The band intensities were quantified with ImageJ. The inhibition of auto-palmitoylation by compounds was normalized to DMSO. The IC50 curves were plotted with GraphPad Prism6.

Pierce™ Streptavidin-coated, high capacity, black 384-well plates were obtained from Life Technologies (Grand Island, NY), alkyne-palmitoyl-CoA was obtained from Cayman Chemicals (Ann Arbor, MI), MDCK (Madin-Darby canine kidney epithelial) cells (ATCC^® CCL-34™) were obtained from ATCC (Manassas, VA), the fluorogenic probe CalFluor 488 was synthesized by the Bertozzi lab; palmitoyl CoA, 2-bromopalmitate, 2-(N–morpholino) ethanesulfonic acid (MES), and tris(2-carboxyethyl)phosphine (TCEP) were obtained from Sigma Aldrich (St. Louis, MO); 3 [tris (3-hydroxypropyltriazolylmethyl) amine (THPTA), maleimide–PEG₄–alkyne, and biotin–PEG₄–alkyne were obtained from Bioconjugate Technologies (Scottsdale, AZ).

Recombinant His₆-TEAD2 (500 ng) protein was pretreated with MGH-CP compounds at indicated doses for 0.5 h followed by incubation with the 1 μM of alkyne palmitoyl-CoA (Cayman Chemical) for 0.5 h in 50 mM MES buffer (pH 6.4). Click reaction was performed as described previously^{65 (link)}. Briefly, CuSO₄/TBTA/TCEP/Biotin-Azide master mix was added into 50 μL protein/palmitoyl-CoA reaction buffer, making the final concentration CuSO₄ 100 µM, TBTA 10 µM, TCEP 100 µM and Biotin-Azide 10 µM. Samples were incubated for 1 h at room temperature, followed by SDS-PAGE analysis. Biotinylated TEAD protein was detected by streptavidin-HRP. Band intensities obtained from streptavidin blots were quantified using ImageJ (NIH).