Expression vectors for INPP5E and its mutants used in this study are listed in Table S1 ; some of them were constructed in our previous study (Nozaki et al., 2017 (link)); the INPP5E and SSTR3 cDNAs were originally provided by Tamotsu Yoshimori (Osaka University) (Hasegawa et al., 2016 (link)) and Yumiko Saito (Hiroshima University) (Nagata et al., 2013 (link)), respectively. Plasmids containing FKBP and FRB sequences were kind gifts from Takanari Inoue (Johns Hopkins University) (Komatsu et al., 2010 (link)). Point and deletion mutants of INPP5E and EGFP-INPP5E with the N-terminal FKBP sequence, and SSTR3-mChe with the C-terminal FRB sequence, were constructed using the SLiCE cloning method (Motohashi, 2015 (link)). Packaging plasmids for the production of lentiviral vectors were kind gifts from Peter McPherson (McGill University) (Thomas et al., 2009 (link)). Antibodies used in this study are listed in Table S2 . SAG, polyethylenimine Max, and rapamycin were purchased from Enzo Life Sciences, Polysciences, and LC Laboratories, respectively.
Polyethylenimine max
Manufactured by Enzo Life Sciences
Polyethylenimine Max is a cationic polymer used for transfection of cells. It is effective in delivering nucleic acids, such as plasmids or siRNA, into a variety of mammalian cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Glutathione sepharose 4b beads, by GE Healthcare (2 mentions)
Rapamycin, by Enzo Life Sciences (1 mentions)
Hek293t cells rbc2202, by RIKEN BioResource Center (1 mentions)
Htert rpe1 cells, by RIKEN BioResource Center (1 mentions)
Peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Gtu 88, by Merck Group (1 mentions)
Pm005, by MBL Life Science (1 mentions)
Ab38686, by Abcam (1 mentions)
5 protocols using polyethylenimine max
1
INPP5E and SSTR3 Constructs for Study
2
Expression Vectors and Antibodies for IFT Complexes
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Expression vectors for subunits of the IFT-A and IFT-B complexes and heterotrimeric kinesin-II used in this study are listed in Supplemental Table S1; most of them were constructed in our previous studies (Katoh et al., 2016 (link); Hirano et al., 2017 (link); Funabashi et al., 2018 (link)). The antibodies used in this study are listed in Supplemental Table S2. GST-tagged anti-GFP Nb (Katoh et al., 2015 (link)) and anti-mCherry Nb (the LaM-2 version) (Ishida et al., 2021 (link)) prebound to Glutathione Sepharose 4B beads (GE Healthcare) were prepared as described previously. SAG and Polyethylenimine Max were purchased from Enzo Life Sciences and Polysciences, respectively. HEK293T cells (RBC2202) and hTERT-RPE1 cells (CRL-4000) were obtained from the RIKEN BioResource Research Center and the American Type Culture Collection, respectively.
3
Antibody Immunostaining Protocol
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The following antibodies were obtained from the indicated vendors: monoclonal mouse anti–Ac-α-tubulin (1:500; 6-11B-1; Sigma-Aldrich); monoclonal rabbit anti–Ac-α-tubulin (1:300; D20G3; Cell Signaling Technology); monoclonal mouse anti–γ-tubulin (1:1000; GTU88; Sigma-Aldrich); polyclonal rabbit anti-Arl13b (1:1000; 17711-1-AP; ProteinTech); polyclonal rabbit anti-IFT88 (1:200; 13967-1-AP; ProteinTech); polyclonal rabbit anti-Smo (1:200; ab38686; Abcam); monoclonal mouse anti-GFP (1:1000; JL-8; BD Biosciences); polyclonal rabbit anti-RFP (1:1000; PM005; MBL Life Science); Alexa Fluor–conjugated secondary antibodies (Molecular Probes); and peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). SAG and Polyethylenimine Max were purchased from Enzo Life Sciences and Polysciences, respectively. GST-tagged anti-GFP Nb prebound to glutathione–Sepharose 4B beads were prepared as described previously (Katoh et al., 2015 (link)).
4
Isolation of GFP-tagged proteins
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The antibodies used in this study are listed in Supplemental Table S4. GST-tagged anti-GFP Nb prebound to glutathione–Sepharose 4B beads was prepared as described previously (Katoh et al., 2015 (link)). SAG and Polyethylenimine Max were purchased from Enzo Life Sciences and Polysciences, respectively.
5
Dynein-2 Subunit Expression and Antibodies
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Expression vectors for DYNC2LI1 and other dynein-2 subunits used in this study are listed in Table S2 ; some of them were constructed in our previous studies18 (link),19 (link). The antibodies used in this study are listed in Table S3 . GST-tagged anti-GFP Nb and anti-mChe Nb (LaM-2 version) prebound to glutathione–Sepharose 4B beads (GE Healthcare) were prepared as described previously33 (link),46 (link),47 (link). SAG and polyethylenimine Max were purchased from Enzo Life Sciences and Polysciences, respectively.
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