Cell lysis and immunoblotting were performed as described previously (34 (link)). For phosphatase treatment, cell lysates were incubated with alkaline phosphatase (E. coli C75) (TAKARA BIO INC.) in the reaction buffer (50 mM Tris-HCl (pH8.8), 1 mM MgCl2) for 30 min at 60 °C. Actin stress fiber formation was detected by staining with Rhodamine-conjugated phalloidin (Cytoskeleton), as described in Motizuki et al. (62 (link)). Chamber migration assay was performed as described in Motizuki et al. and Lee et al. (11 , 33 (link)). Quantitative real-time PCR was performed as described in Itoh et al. (63 (link)). Primer sequences are shown in Table S1 .
E coli c75
Manufactured by Takara Bio
E. coli C75 is a strain of Escherichia coli bacteria commonly used in laboratory settings. It is a non-pathogenic organism that serves as a model organism for various research and experimental purposes.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using e coli c75
1
Cell Lysis, Immunoblotting, and Migration Assay
2
Generating CRISPR-dCas9 Expression Plasmids
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To construct MSCV expression plasmids expressing 3xFLAG-Sp-dCas9, the pMSCVneo, pMSCVhyg, and pMSCVpuro vectors (631461, Takara Bio, Kusatsu, Japan) were digested with BglII (1021A, Takara Bio) and Xho I (1094A, Takara Bio). After a blunting reaction, the plasmids were treated with bacterial alkaline phosphatase (E. coli C75) (2120A, Takara Bio). The cleaved vectors were puri ed by agarose gel electrophoresis and ligated with the coding sequence of 3xFLAG-Sp-dCas9, which was isolated from 3xFLAG-dCas9/pMXs-puro (Addgene (Watertown, MA, USA) #51240) (13) by digestion with Pac I (R0547, New England Biolabs, Ipswich, MA, USA) and Not I (1166A, Takara Bio).
To construct gRNA-hIRF-1 #12/pSIR-hCD2 (Addgene #135392), pSIR-hCD2 (Addgene #51143) (13) was digested with EcoR I (1040A, Takara Bio) and treated with bacterial alkaline phosphatase. The cleaved vector was puri ed by agarose gel electrophoresis and ligated with the gBlock targeting the interferon (IFN) regulatory factor (IRF)-1 gene promoter, which was isolated from gRNA-hIRF-1 #12 (Addgene #61079) (1) by digestion with EcoR I.
The Addgene plasmid # of the newly made constructs are as follows: 3xFLAG-Sp-dCas9/pMSCVneo: 134982; 3xFLAG-Sp-dCas9/pMSCVhyg: 134323; 3xFLAG-Sp-dCas9/pMSCVpuro: 134983; gRNA-hIRF-1 #12/pSIR-hCD2: 135392.
To construct gRNA-hIRF-1 #12/pSIR-hCD2 (Addgene #135392), pSIR-hCD2 (Addgene #51143) (13) was digested with EcoR I (1040A, Takara Bio) and treated with bacterial alkaline phosphatase. The cleaved vector was puri ed by agarose gel electrophoresis and ligated with the gBlock targeting the interferon (IFN) regulatory factor (IRF)-1 gene promoter, which was isolated from gRNA-hIRF-1 #12 (Addgene #61079) (1) by digestion with EcoR I.
The Addgene plasmid # of the newly made constructs are as follows: 3xFLAG-Sp-dCas9/pMSCVneo: 134982; 3xFLAG-Sp-dCas9/pMSCVhyg: 134323; 3xFLAG-Sp-dCas9/pMSCVpuro: 134983; gRNA-hIRF-1 #12/pSIR-hCD2: 135392.
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