To create the SaCas9/anti-HERV-KenvgRNA constructs, we used the existing pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::Bsa1-sgRNA plasmid (Addgene, #61591), consisting of a Staphylococcus aureus-derived SaCas9/gRNA system, adapted for use in mammalian cells [33 (link)]. Protospacer regions corresponding to selected target sites were ordered, as a pair of 5′-G(N22)-3′ complementary oligonucleotides, containing Bsa1 overhangs at their respective 5′ ends. After annealing and phosphorylation by T4 polynucleotide kinase (New England Biolabs), double stranded protospacers were ligated into the Bsa1-digested pX601 backbone plasmid, which was dephosphorylated with calf intestine phosphatase (New England Biolabs, #M0290S). Bacterial clones were screened by PCR for the presence of gRNA protospacer inserts, using the top forward oligonucleotide of each annealed gRNA, in combination with reverse primer specific for the gRNA scaffold segment of U6-gRNA cassette. Successful clones were further verified by sequencing, using the same reverse primer.
Calf intestine phosphatase
Manufactured by New England Biolabs
Calf intestine phosphatase (CIP) is an enzyme that catalyzes the removal of 5' phosphate groups from DNA, RNA, and other phosphorylated molecules. It is commonly used in molecular biology applications to prepare DNA fragments for ligation or to remove 5' phosphates from dephosphorylated vectors prior to ligation.
Automatically generated - may contain errors
Lab products found in correlation
T4 polynucleotide kinase, by New England Biolabs (1 mentions)
Anti ha antibody, by Abcam (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Thermo Fisher Scientific (1 mentions)
Vinculin, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Lambda protein phosphatase lambda pp, by New England Biolabs (1 mentions)
Udp disodium salt, by Biosynth (1 mentions)
15hcm β d glucoside, by BASF (1 mentions)
Cutsmart buffer, by New England Biolabs (1 mentions)
Effectene transfection reagent, by Qiagen (1 mentions)
Anti dfoxo, by Cosmo Bio (1 mentions)
Hrp linked antibody, by Cosmo Bio (1 mentions)
6 protocols using calf intestine phosphatase
1
Generating SaCas9/anti-HERV-K Constructs
2
BIK1 Phosphorylation Analysis in Flg22-Treated Protoplasts
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BIK1 phosphorylation was determined by a BIK1 mobility shift as previously described (Lu et al., 2010 (link)). Protoplasts were transfected with BIK1-HA construct and treated with 1 μM flg22 for 15 min. Total proteins were extracted using an extraction buffer [10 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Triton X-100, and 1x protease inhibitor cocktail (Roche)], and treated with calf intestine phosphatase (New England Biolabs) to trigger dephosphorylation. Extracted proteins were subjected to boiling in 2X Laemmli sample buffer (100 mM Tris-HCl, pH 6.8, 10% SDS, 30% glycerol, 5% β-mercaptoethanol, and 0.02% bromophenol blue), separated by SDS-PAGE, and visualized by immunoblotting with anti-HA antibody (Abcam).
3
Invadopodia and Cytoskeletal Protein Assays
4
Glucosylation and Phosphatase Analysis
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Chemicals were from Carl Roth
(Karlsruhe, Germany) and Sigma-Aldrich (Vienna, Austria). 4MU (3 , Figure 1 ; ≥98%), 4NP (4 , Figure 1 ; ≥99%) and 4NP-β-d -glucoside (≥98%) were from Sigma-Aldrich. 4MU-β-d -glucoside, UDP disodium salt, and UDP-glucose disodium salt
were from Carbosynth (Compton, U.K.). 15HCM (1 , Figure 1 ; ≥99.5%)
and 15HCM β-d -glucoside (5 , Figure 1 ; ≥99%) were provided
by BASF. Note: 15HCM is a racemic mixture of the (3R, 5R, 6S) and (3S, 5S, 6R) forms (Figure 1 ). 15HCM and the glycosylated
derivatives thereof are toxic and irritant. Proper care was taken
in their handling. Calf intestine phosphatase (CIP, 10 000
U/mL) was from New England Biolabs (Frankfurt am Main, Germany).
(Karlsruhe, Germany) and Sigma-Aldrich (Vienna, Austria). 4MU (
were from Carbosynth (Compton, U.K.). 15HCM (
and 15HCM β-
by BASF. Note: 15HCM is a racemic mixture of the (3R, 5R, 6S) and (3S, 5S, 6R) forms (
derivatives thereof are toxic and irritant. Proper care was taken
in their handling. Calf intestine phosphatase (CIP, 10 000
U/mL) was from New England Biolabs (Frankfurt am Main, Germany).
5
Phosphatase Treatment of HeLa Cell Lysate
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HeLa cells (5 × 106), treated as indicated, were lysed in 750 µL lysis buffer (containing 20 mM Tris/HCl, pH 8.0, 50 mM KCl, 2 mM MgCl2, [v/v] 0.2% NP-40) containing protease inhibitors; 40 µL lysate were incubated with CutSmart Buffer (1× final, New England Biolabs) and 15 units calf intestine phosphatase (CIP, 10 units/µL, New England Biolabs) for 45 min at 37°C. Reactions were stopped by adding 2× SDS-PAGE loading buffer and denaturation for 5 min at 95°C.
6
Drosophila FoxO Interaction and Phosphorylation
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Drosophila S2R+ cells were cultured in Corning Insectagro DS2 with 10% FBS (HyClone). Plasmids pUAST-Flag-dFoxO, pUAST-3HA-Pelle and Actin-GAL4 were used for co-transfection as indicated using Effectene Transfection Reagent (QIAGEN). 48 hours after transfection, cells were lysed in RIPA buffer (CST) with PMSF and proceeded with the standard western blot and co-immunoprecipitation protocols. Proteins were probed or immunoprecipitated with following antibodies: Flag-Tag antibody [3A6] (CMCTAG), HA-Tag (C29F4) Rabbit mAb (CST), Anti dFoxO (Cosmo Bio), Anti-rabbit IgG, HRP-linked Antibody (CST), Anti-mouse IgG, HRP-linked Antibody (CST). For Phosphatase Treatment, cell extracts were incubated with calf intestine phosphatase (CIP) (New England BioLabs) for 40 mins at 37°C.
+ Expand
Drosophila S2R+ cells were cultured in Corning Insectagro DS2 with 10% FBS (HyClone). Plasmids pUAST-Flag-dFoxO, pUAST-3HA-Pelle and Actin-GAL4 were used for co-transfection as indicated using Effectene Transfection Reagent (QIAGEN). 48 hours after transfection, cells were lysed in RIPA buffer (CST) with PMSF and proceeded with the standard western blot and co-immunoprecipitation protocols. Proteins were probed or immunoprecipitated with following antibodies: Flag-Tag antibody [3A6] (CMCTAG), HA-Tag (C29F4) Rabbit mAb (CST), Anti dFoxO (Cosmo Bio), Anti-rabbit IgG, HRP-linked Antibody (CST), Anti-mouse IgG, HRP-linked Antibody (CST). For Phosphatase Treatment, cell extracts were incubated with calf intestine phosphatase (CIP) (New England BioLabs) for 40 mins at 37°C.
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