Gfp p4m sidm

Manufactured by Addgene

GFP-P4M-SidM is a fluorescent protein construct that can be used to visualize phosphatidylinositol 4-phosphate (PI4P) in live cells. It consists of green fluorescent protein (GFP) fused to the PI4P-binding domain of the Legionella protein SidM.

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3 protocols using gfp p4m sidm

Visualizing Phosphoinositide Dynamics

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All salts were from Sigma-Aldrich. PAO was from Sigma-Aldrich, and wortmannin and PIK93 were from TOCRIS Bioscience. The following plasmids were used: GFP-PH-OSBP (Balla et al., 2005 (link); gift from Tamas Balla, National Institutes of Health, Bethesda, MD), GFP-PHx2-OSH2 (Stefan et al., 2011 (link); plasmid 36095; Addgene), GFP-P4M-SidM (Hammond et al., 2014 (link); plasmid 51472; Addgene), mRFP-PH-PLCδ1, mRFP-PH-Akt, GFP-Sac2 (Nakatsu et al., 2015 (link); gift from Pietro DeCamilli, Yale University, New Haven, CT), GFP-Rab27a, GFP-Rab3a, NPY-GFP, NPY-mCherry, VAMP2-pHluorin (all gifts from Sebastian Barg, Uppsala University, Uppsala, Sweden), Lamp1-RFP (Sherer et al., 2003 (link); plasmid 1817; Addgene), GFP-Rab5, GFP-EEA1 (gifts from Pietro DeCamilli), CIBN-CAAX (Idevall-Hagren et al., 2012 (link)), GFP-CRY2-5ptase_OCRL (Idevall-Hagren et al., 2012 (link)), GFP-CRY2-iSH2 (Idevall-Hagren et al., 2012 (link)), R-GECO1 (Zhao et al., 2011 (link); plasmid 32444; Addgene), GFP-Lact-C2 (Yeung et al., 2008 (link); plasmid 22853; Addgene), EGFP-Rab4a (Rzomp et al., 2003 (link); plasmid 49434; Addgene), GFP-Rab10 (Huckaba et al., 2011 (link); plasmid 31737; Addgene), Granuphilin-GFP (Gálvez-Santisteban et al., 2012 (link); plasmid 40032; Addgene), and GFP-2xFYVE_EEA1 (Petiot et al., 2003 (link)).

Nguyen P.M., Gandasi N.R., Xie B., Sugahara S., Xu Y, & Idevall-Hagren O. (2019). The PI(4)P phosphatase Sac2 controls insulin granule docking and release. The Journal of Cell Biology, 218(11), 3714-3729.

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Plasmid Transfection and Infection in RAW264.7 Cells

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The following probes GFP-P4M-SidM (Addgene plasmid # 51469); GFP-P4M-SidMx2(Addgene plasmid #51472); PH-Akt-GFP (Addgene plasmid # 51465); PH-Akt(R25C)-GFP (Addgene plasmid # 51466) PH-PLCD1-GFP (Addgene plasmid # 51407); PH-PLCD1(R40L)-GFP (Addgene plasmid # 51408); PH-Btk-GFP (Addgene plasmid # 51463); PH-Btk(R28C)-GFP (Addgene plasmid # 51464) were gifts from Tamas Balla; GFP-EEA1 wt (Addgene plasmid # 42307) was a gift from Silvia Corvera; pBGPa-CMV-GFP-OSBP PH domain (Addgene plasmid # 58840) was a gift from Tim Levine & Sean Munro. mKate2-P2A-APEX2-TAPP1-PH (Addgene Plasmids #67662) was a gift was a gift from Rob Parton. Plasmids were isolated and test digested for confirmation. They were then introduced into RAW264.7 by nucleofection using reagents from Mirus Bio (Madison, Wisconsin). After seeding transfected cells and overnight culture, they were infected. Infection of transfectants were evaluated after 2 or 24hr.

Zhang N., Prasad S., Despointes C.E., Young J, & Kima P.E. (2018). Leishmania parasitophorous vacuole membranes display phosphoinositides that create conditions for continuous Akt activation and a target for miltefosine in Leishmania infections. Cellular microbiology, 20(11), e12889.

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Cloning and Tagging of ER and Membrane Proteins

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Tex2 (NM_018469.5), TMEM55A (NM_018710.3), TMEM55B (NM_001100814.3), ALT1, ARL6IP1, E-Syt1, RTN4, and Reep5 were cloned from HeLa cDNA. The ORFs of Tex2, TMEM55B, TMEM55A, ALT1, PI4KIIα, PI4KII β, and E-syt1 were cloned into mEGFP-C1 between the BglII and SacI or Halo-C1 vector between the SacI and BamHI. The ORFs of Reep5, RTN4, and MGAT2 were cloned between NheI and SacI of Halo-N1. GST-TMEM55B was cloned into PGEX-2T vector between the BamHI and EcoRI. 14xHis-NEDD8-Tex2-SMP was cloned into 14xHis-NEDD8 vector between the BamHI and HindIII. ER-tagRFP and Lamp1-mCh were previously described (Gao et al., 2022 (link); Ji et al., 2017 (link)). GFP-Lact-C2 (22852; Addgene), CFP-C1-PLCδ-PH (21262; Addgene), GFP-P4M-SidM (51469; Addgene), mApple-Lamp1-phLuorin-N-8 (54918; Addgene), and PMRX–IP–GFP–LC3–RFP (84573; Addgene) were purchased from Addgene.

Du Y., Chang W., Gao L., Deng L, & Ji W.K. (2023). Tex2 is required for lysosomal functions at TMEM55-dependent ER membrane contact sites. The Journal of Cell Biology, 222(4), e202205133.

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