alkynyl palmitic acid  (Click Chemistry Tools)

Journal: mBio

Article Title: Plasmodium berghei IMC1j interacts with δ-tubulin to orchestrate subpellicular microtubule organization in ookinetes

doi: 10.1128/mbio.02669-25

Figure Lengend Snippet: Click chemistry assay of PbIMC1j protein in schizonts and ookinetes. ( A ) The localization of the palmitoylated PbIMC1j protein in the schizont (SZ) and ookinete (Ook) stages was identified using click chemistry. Parasites were metabolically labeled with alkynyl palmitic acid (Alk-C16) and subsequently stained with Alexa Fluor 488-conjugated streptavidin (Strepv, green), anti-MSP1 serum or anti-P25 serum (cyan), and anti-HA mAb (red). Scale bars: 5 µm. ( B ) Western blot analysis of palmitoylated PbIMC1j proteins in schizonts (left panel) and ookinetes (right panel). The Alk-C16 labeled (+) proteins were detected using anti-HA mAb. S, immunoprecipitation (IP) supernatant; P, IP elution. The arrowhead indicates the PbIMC1j-HA protein. ( C ) IFA of PbIMC1j HA- glmS parasites at the schizont stage (SZ) after treatment without (−) or with 100 µM of 2 BP (+). Scale bars: 5 µm. ( D ) PbIMC1j-HA localization in ookinetes treated without (−) or with (+) 100 µM of 2 BP. Scale bars: 5 µm. ( E ) Solubility of the PbIMC1j-HA in schizonts following 2 BP treatment. Sequential extraction was performed with freeze-thaw ( F ), 1% Triton X-100 (T), and 2% SDS (S). GAPDH (soluble) and GAPM2 (membrane) serve as fractionation controls. ( F ) Solubility profile of PbIMC1j-HA protein in ookinetes after 2 BP treatment. Extraction conditions as in ( D ). All panels are representative of three biological replicates.

Article Snippet: For the click chemistry assay , schizonts or ookinetes were resuspended in a culture medium containing 100 μM of alkynyl palmitic acid (Alk-C16, TargetMol, MA, USA) and then incubated at 37°C for 4 hours.

Techniques: Metabolic Labelling, Labeling, Staining, Western Blot, Immunoprecipitation, Solubility, Extraction, Membrane, Fractionation

Journal: Journal of Virology

Article Title: Palmitoylation enhances the stability of porcine epidemic diarrhea virus spike protein by antagonizing its degradation via chaperone-mediated autophagy to facilitate viral proliferation

doi: 10.1128/jvi.00347-25

Figure Lengend Snippet: PEDV S protein is palmitoylated through thioester bonds. ( A ) A schematic for detecting protein palmitoylation by IP, click chemistry, and streptavidin pull-down assays. ( B ) HEK-293T cells were transfected with the plasmid encoding PEDV S-Flag or Flag-tagged empty vector for 24 h and metabolically labeled with alkynyl PA or DMSO as a control for 8 h. The supernatant of WCL was immunoprecipitated using anti-Flag magnetic beads. The beads were subjected to click chemistry, and treatment with or without HAM, followed by elution with glycine-HCl. The eluate was pulled down with streptavidin beads, and the precipitated proteins were analyzed by IB. ( C ) HEK-293T cells were transfected with the plasmid encoding PEDV S-Flag or Flag-tagged empty vector. The cells were treated with or without 40 µM 2-BP for 24 h and then metabolically labeled with Alkynyl PA or DMSO as a control for 8 h. Subsequent assays were performed as described for panel B. ( D ) Prediction of palmitoylation sites in PEDV S proteins. ( E ) HEK-293T cells were transfected with the plasmid encoding PEDV S-Flag, S-ΔCRR, or S-CA for 24 h. Subsequent assays were performed as described for panel B. The mean gray values of S protein were quantified using ImageJ software.

Article Snippet: CHX (Cat. No. HY-12320), 3-MA (Cat. No. HY-19312), carfilzomib (Cat. No. HY-10455), NH 4 Cl (Cat. No. HY-Y1269), CQ (Cat. No. HY-17589A), Alkynyl PA (Cat. No. HY-W040304), Tris(benzyltriazolylmethyl)amine (TBTA; Cat. No. HY-116677), biotin-azide (Cat. No. HY-129832), streptavidin magnetic beads (Cat. No. HY-K0208), and Protein G magnetic beads (Cat. No. HY-K0204) were purchased from MedchemExpress.

Techniques: Transfection, Plasmid Preparation, Metabolic Labelling, Labeling, Control, Immunoprecipitation, Magnetic Beads, Software

Journal: Journal of Virology

Article Title: Palmitoylation enhances the stability of porcine epidemic diarrhea virus spike protein by antagonizing its degradation via chaperone-mediated autophagy to facilitate viral proliferation

doi: 10.1128/jvi.00347-25

Figure Lengend Snippet: Palmitoylation of PEDV S protein was mediated by ZDHHC5 during infection. ( A ) Vero cells were infected with PEDV at a multiplicity of infection (MOI) of 0.05 for 12 h and metabolically labeled with alkynyl PA or DMSO as a control for 8 h. The supernatant of WCL was immunoprecipitated with 10 µg anti-PEDV S mAb and 100 µL protein G magnetic beads. The beads were subjected to click chemistry, and treatment with or without HAM, followed by elution with glycine-HCl. The eluate was pulled down with streptavidin beads, and the precipitated proteins were analyzed by IB. ( B ) Vero cells were infected with PEDV at 0.05 MOI for 12 h. The cells were treated with or without 6 µM 2-BP and metabolically labeled with Alkynyl PA or DMSO as a control for 8 h. Subsequent assays were performed as described for panel A. ( C ) PEDV was propagated in Vero cells at 0.05 MOI for 12 h and metabolically labeled with 50 µM alkynyl PA for 8 h. The mature virions were purified from the culture supernatant by sucrose gradient supercentrifugation and detected by SDS-PAGE. Subsequent assays were performed as described for panel A. ( D ) Vero cells were infected with PEDV at 0.25 MOI for 8 h and then lysed. Using S protein as bait, the precipitated proteins were analyzed by IB. Isotype IgG antibody was used as a negative control. ( E ) Vero cells were infected with PEDV at 0.25 MOI for 8 h. Then, PEDV S protein, ZDHHC5, and calreticulin were visualized with the specific primary and secondary antibodies. Subsequent assays were performed as described for . ( F ) Vero cells were transfected with si- ZDHHC5 or si-NC for 24 h and were then infected with PEDV at 0.05 MOI for 12 h. Subsequent assays were performed as described for panel A. The mean gray values of S protein were quantified using ImageJ software.

Article Snippet: CHX (Cat. No. HY-12320), 3-MA (Cat. No. HY-19312), carfilzomib (Cat. No. HY-10455), NH 4 Cl (Cat. No. HY-Y1269), CQ (Cat. No. HY-17589A), Alkynyl PA (Cat. No. HY-W040304), Tris(benzyltriazolylmethyl)amine (TBTA; Cat. No. HY-116677), biotin-azide (Cat. No. HY-129832), streptavidin magnetic beads (Cat. No. HY-K0208), and Protein G magnetic beads (Cat. No. HY-K0204) were purchased from MedchemExpress.

Techniques: Infection, Metabolic Labelling, Labeling, Control, Immunoprecipitation, Magnetic Beads, Purification, SDS Page, Negative Control, Transfection, Software