2 bromopalmitate

2-Bromopalmitate (or 2-bromohexadecanoic acid, Sigma-Aldrich, Cat no. 238422) was dissolved in dimethyl sulfoxide (Sigma-Aldrich, Cat no. D2650) for making concentrated stock solution. Stock solution was diluted in the appropriate culture medium to a final concentration of 10 μM. Cells were incubated with either 2-Bromopalmitate-containing medium or control medium (the same quantity of solvent diluted in culture medium) overnight, in a humidified chamber at 37°C with 5% CO₂. The use of ethanol as a solvent worked equally well. No particular toxicity on cultured cells was observed following incubation.

Progesterone and R5020 were obtained from Perkin-Elmer. Lipofectamine 2000 Transfection reagent was from Thermo Fisher. 2-Bromopalmitate was acquired from Sigma. pcDNA3.1 ( +) SNAP-GFP-PRb wt and pcDNA3.1 ( +) SNAP-GFP-PRb C820A plasmids were generated from pTET-Splice SNAP-GFP-PRb plasmid kindly given by Gordon Hager, subcloned into pcDNA3.1( +) backbone. Mutagenesis of the palmitoylation site (aa Cys 820 into Ala) was done by GenScript Biotech (Netherlands) B.V. pAGEMMTVLu(MMTV-luc) was used to assess expression of the Progesterone responsive promoter MMTV in U2OS cells.

2-Bromopalmitate and S-Methyl methanethiosulfonate (MMTS) were from Sigma. All other chemicals were from ThermoFisher Scientific (Waltham, MA) and were of the highest reagent grade.

Studies of protein palmitoylation were performed according to detailed published protocols[95 (link)–97 (link)]. In brief, transfected cells were treated for 1 h with the alk-16 (20 uM) chemical reporter of protein palmitoylation. Proteins of interest were immunoprecipitated from cell lysates and reacted via the copper(I)-catalized azide alkyne cycloaddition reaction (“click chemistry”) with azido-rhodamine (kindly provided by Dr. Howard Hang of the Rockefeller University) for visualization of protein acylation via fluorescence gel scanning on a Typhoon 9400 (Amersham) fluorescence imager. Western blotting of the samples provided controls for loading and sample inputs. For inhibition of protein palmitoylation, transfected cells were treated for 24 h with 100 uM 2-bromopalmitate (Sigma).

Lipofectamine 2000, Lipofectamine RNAiMax, sodium dedocyl sulfate (SDS) solution, L-azidohomoalanine (L-AHA), Alexa Fluor 488-azide (AF488-az), Alexa Fluor 647-alkyne (AF647-alk), TRIzol reagent, and Prolong Gold Antifade Mountant with DAPI were purchased from Life Technologies (Burlington, ON). X-tremeGENE 9 was purchased from Roche (Indianapolis, IN). Palmostatin B was purchased from Merck Scientific (Billerica, MA). Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), Triton-X 100 (TX-100), sodium deoxycholate, CuSO₄, palmitic acid, and 2-bromopalmitate were obtained from Sigma-Aldrich (St. Louis, MO). 17-ODYA, C75, WWL70, and RHC-80267 were purchased from Cayman Chemical (Ann Arbor, MI). HDFP, C83, and C115 were gifts from Dr. Brent Martin (University of Michigan), and FP-rhodamine was generously provided by Dr. Benjamin Cravatt (Scripps Institute).

T47D cells were exposed to 10 μM 2-bromopalmitate (Sigma) for 6 h prior to hormone induction.