Palmitoylation targets the Calcineurin phosphatase to the Phosphatidylinositol 4-kinase complex at the plasma membrane
Description
This dataset is associated with the publication Ulengin-Talkish, I. et. al., 2021. This manuscript is currently under revision in Nature Communications. In this study, we elucidate a new signaling pathway regulated by the Ca2+-activated phosphatase, calcineurin (CN) by examining the understudied isoform CNAB1. Using multiple cell based biochemical assays including pulse-chase experiments with metabolic analog of palmitate and click chemistry, we show that CNAB1 is dynamically palmitoylated at two sites unique to its C-terminal tail. This dynamic palmitoylation, regulated by the ABHD17A depalmitoylase, confers unique localization to the plasma membrane (PM) and the golgi, substrate specificity and regulation to this isoform. We further uncover the PM-associated phosphatidylinositol 4-kinase complex to be a regulatory target of this isoform through which CN regulates PI4P production at the PM during signaling from the type-3 muscarinic receptor. This dataset specifically contains indirect immunofluorescence experiments done in COS-7 cells, which were used for the analysis shown in figures 1, 2 and supplementary figure 1. These multichannel images were acquired on a single z-plane on LionheartTM FX automated widefield microscope with a 20X Plan Fluorite WD 6.6 NP 0.45 objective.
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COS-7 cells were grown on 12 mm, #1.5H glass coverslips (ThorLabs). 24 h post-transfection, cells were washed with 1X PBS and fixed in 4% paraformaldehyde (PFA) solution (diluted from 16% PFA, Electron Microscopy Sciences) in PBS for 15 min. Cells were washed thrice with PBS and permeabilized for 5 min in block buffer (1x PBS with 0.2 M Glycine, 2.5% FBS) with 0.1% Triton X-100. Cells were then incubated in block buffer without detergent for 30 min. Coverslips were incubated with primary antibodies diluted in block buffer (without detergent) for 1 h, washed multiple times with 1x PBS followed by incubation with secondary antibodies for 1 h at room temperature. Coverslips were washed again and mounted using Prolong Diamond Antifade mountant (Thermo Fisher). Images were acquired on a single z-plane on LionheartTM FX automated widefield microscope with a 20X Plan Fluorite WD 6.6 NP 0.45 objective. For Figure. 1e and Supplementary Figure. 1e, primary antibodies used: mouse anti-FLAG, M2 (1:500, Sigma-Aldrich, F1804) and rabbit anti-GM130, D6B1 (1:400, Cell Signaling Technologies, 12480). Secondary antibodies used: Anti-mouse Alexa Fluor 647 (1:500, Invitrogen) and anti-rabbit Brilliant Violet 421 (1:100, Biolegend). YFP (500 nm), Texas Red (590 nm) and DAPI (350 nm) filter cubes were used to image Venus, FLAG and GM130 respectively. For Figure. 2f: GFP (465 nm), Texas Red (590 nm) and DAPI filter cubes were used to image GFP, FLAG and GM130, respectively