Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2016 Oct 4;6:34479. doi: 10.1038/srep34479

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

Copyright © 2016, The Author(s)

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

PMC Copyright notice

(A) Autoradiograph of in vitro phosphorylation samples (upper panel) as well as coomassie stained loading (lower panel) are shown. AMA1 variants displaying either all (AMA1_WT), none (AMA1_PM) or only single phosphorylation sites (AMA1_S610, AMA1_T612, AMA1_T613) were incubated with schizont material in the presence of cAMP and ³²P-γ-ATP. (B) Phosphorylation of PKA pre-phosphorylated AMA1_WT and mutants AMA1_PM and AMA1_S610/T613. Recombinant proteins were first incubated with PKA in the presence of non-labeled ATP. Subsequently, these pre-treated AMA1 were incubated with either PKA or a schizont extract in the presence of ³²P-γ-ATP. AMA1_PM was used as a control. SDS-PAGE and radiograph of in vitro phosphorylation samples (upper panel) as well as coomassie stained loading (lower panel) are shown. (C) In vitro phosphorylation of AMA1-GST fusion variants (AMA1_WT, AMA1_S610, AMA1_S610/T613) either as unphosphorylated (-) or PKA-pre-phosphorylated (+) substrates in the presence of recombinant PfGSK3β and ³²P-γ-ATP or without the addition of PfGSK3β (c). (D) Signal intensities were quantified. Error bars correspond to standard deviation of two independent experiments done in triplicates. (E) Sandwich ELISA demonstrating 5v-induced inhibition of native AMA1 threonine phosphorylation. Parasites were treated with 5v and a rabbit anti-PfAMA1 antibody was used to capture PfAMA1 from culture lysates. Threonine phosphorylation was detected in an ELISA format using a mouse anti-phosphothreonine antibody. Histograms were generated after normalizing against uninfected (0%, background) and untreated (100%) culture signals. Phosphatase-treatment was used to denote zero phosphorylation and chloroquine treatment was used to exclude parasite growth arrest as a cause for reduced phosphorylation. (F) The PfPKA inhibitor H89, weakly inhibited egress and strongly inhibited invasion in P. falciparum reporter parasites transfected with secreted Nanoluciferase. Luciferase activity in relative light units was measured and dose-response curves were plotted for egress (dashed line) and invasion (solid line) after normalizing against uninfected (0%) and untreated (100%) culture sample signals. Two biological replicates were performed in triplicate; error bars denote one standard deviation. (G) The PfGSK inhibitor 5v strongly inhibits invasion. The experiment was performed as per F.