Ph btk gfp

Manufactured by Addgene

PH-Btk-GFP is a fluorescent protein construct that contains the pleckstrin homology (PH) domain of the Bruton's tyrosine kinase (Btk) protein fused to the green fluorescent protein (GFP). This construct is commonly used to study the localization and dynamics of Btk-PH domain within cells.

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5 protocols using ph btk gfp

Fluorescence Microscopy Plasmid Toolkit

Cited 1 time

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mApple-α-5-Integrin-12 (plasmid #54864; RRID:Addgene_54864; Addgene), mCherry-Clathrin LC-15 (plasmid #55019; RRID:Addgene_55019; Addgene), and mCherry-Rab5a-7 (plasmid #55126; RRID:Addgene_55126; Addgene) were gifts from Michael Davidson (Rizzo et al., 2009 (link)); pcDNA3/hArf1(WT)-mCherry (plasmid #79419; RRID:Addgene_79419; Addgene) was a gift from Kazuhisa Nakayama (Makyio et al., 2012 (link)); CAV1-mCherry (plasmid #27705; RRID:Addgene_27705; Addgene) was a gift from Ari Helenius (Hayer et al., 2010 (link)); and PH-Btk-GFP (plasmid #51463; RRID:Addgene_51463; Addgene) was a gift from Tamas Balla (Várnai and Balla, 1998 (link)).

Le A.H., Yelland T., Paul N.R., Fort L., Nikolaou S., Ismail S, & Machesky L.M. (2021). CYRI-A limits invasive migration through macropinosome formation and integrin uptake regulation. The Journal of Cell Biology, 220(9), e202012114.

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Protein Labeling and Imaging Techniques

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Mutations were introduced into DOC2B-GFP N2 ^{7 (link)}, C2AB-GFP N2 or C2B-GFP N2 ^{6 (link)}, using site-directed mutagenesis PCR. Botulinum toxins BoNT/C and BoNT/E were a generous gift from the laboratory of Prof. Ilana Lotan (Tel-Aviv University). mRFP-syx1A and SNAP25-YFP were a kind gift from Prof. Edward Stuenkel (University of Michigan). R-GECO was a generous gift from the laboratory of Prof. Robert Campbell ^{31 (link)}. Lact-C2-GFP was a gift from Prof. Sergio Grinstein (addgene #22852). For PI manipulations using the rapamycin dimerization system, the PH domain of PLCδ1, PLCδ-PH-GFP (addgene #21179), CF-Inp (addgene #20155), CF-Inp^D281A (addgene #20156) and Lyn11-FRB (addgene #20147) were a gift from Prof. Tobias Meyer. CF-PIPK, CF-PIPKi, mcherry-iSH were a generous gift from the laboratory of Prof. Takanari Inoue (Johns Hopkins University). PLCδ-PH-mKate was a kind gift from Prof. Thomas Martin (University of Wisconsin-Madison). Rab7-FRB (addgene #51613), PH-Akt-GFP (addgene #51465), and PH-Btk-GFP (addgene #51463) were a gift from Prof. Tamas Balla. NPY-mRFP was a kind gift from Prof. Matthijs Verhage (Vrije University).

Michaeli L., Gottfried I., Bykhovskaia M, & Ashery U. (2017). Phosphatidylinositol (4,5)-bisphosphate targets DOC2B to the plasma membrane. Traffic (Copenhagen, Denmark), 18(12), 825-839.

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Signaling Pathways Regulation Analysis

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Recombinant human EGF and human insulin were purchased from Peprotech (AF-100–15) and AbMole (M9194), respectively. The p110α inhibitor A66 (M1819) and the p110β inhibitor TGX221 (M1795) were obtained from AbMole. Cytochalasin D (abs44058674) was from Absin. The protease inhibitor cocktail (04,693,159,001) was purchased from Roche. Anti-AKT (#9272), anti-pAKT (473) (#4060), anti-ERK1/2 (#4376), and anti-pERK1/2 (#4695) antibodies for western blot analysis were purchased from Cell Signaling Technology. Anti-Rab5A(#2143S), anti-AKT(#2920), and anti-insulin receptor β (#23,413) antibodies used for immunofluorescence staining were obtained from Cell Signaling Technology. Anti-RIN1 (16,388-1-AP) and anti-EGFR (18,986-1-AP) antibodies for immunofluorescence staining were obtained from Proteintech. The anti-cortactin (A9518), anti-N-WASP (A2576), anti-p110α (A0265), and anti-p110β (A0928) antibodies for immunofluorescence staining were obtained from ABclonal. Anti-SH3YL1 antibody (NBP1-84,133) for immunofluorescence staining was obtained from Novus. Rhodamine-phalloidin (RM02835) was obtained from Abclonal. The mounting medium with DAPI (ab104139) was from Abcam. The plasmid PH-Btk-GFP (Addgene, #51,463) was used to express GFP-BtkPH.

Sun X., Liu Y., Zhou S., Wang L., Wei J., Hua R., Shen Z, & Yoshida S. (2022). Circular dorsal ruffles disturb the growth factor-induced PI3K-AKT pathway in hepatocellular carcinoma Hep3B cells. Cell Communication and Signaling : CCS, 20, 102.

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Plasmid-Mediated Cell Tracking and Manipulation

Cited 3 times

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Plasmids for tracking PI species were from Addgene. Specifically, mCherry-Clathrin LC-15 (Addgene #55019) [71 (link)]; PLCD1(PH)-mCherry (Addgene #36075) [72 (link)]; PH-PLCD1-GFP (Addgene #51407), PH-PLCD1(R40L)-GFP (Addgene #51408) [73 (link)]; pLentiLifeACT-EGFP BlastR (Addgene #84383) [74 (link)]; PH-Btk-GFP (Addgene #51463), GFP-PH-TAPP1 (Addgene #161985); and pLenti-EGFP-P4M-SidMx2 (Addgene #136997), pLenti-EGFP-2xFYVE (Addgene #136996) were gifts from Ken-Ichi Takemaru. Plasmids were also constructed by Vectorbuilder: VB200917 is a lenti-mCherry-shCLTC containing U6-driven shRNA sequence 5′-CGTGTTCTTGTAACCTTTATT-3′. VB200917 is a lenti-mCherry-shLuciferase containing U6-driven shRNA sequence 5′- ATGTTTACTACACTCGGATAT-3′ and was used to create lentivirus to infect MCF7, 4226, and MPE600 cell lines. Infected cells were sorted for mCherry using FACS as above and tested for engulfment. Human PIK3C2B-GFP fusion overexpression vector is a lentiviral CMV-driven plasmid from VectorBuilder. miRFP-713 (near-infrared fluorescent protein) overexpression vector is a lentiviral EFS-driven plasmid from VectorBuilder. 2xFYVE-domain-mCherry fusion protein is lentiviral plasmid from VectorBuilder. Lentiviral EFS-driven LYN11 mCherry fusion protein construct was also produced by VectorBuilder.

Frey W.D., Anderson A.Y., Lee H., Nguyen J.B., Cowles E.L., Lu H, & Jackson J.G. (2022). Phosphoinositide species and filamentous actin formation mediate engulfment by senescent tumor cells. PLOS Biology, 20(10), e3001858.

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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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