Pcdh cmv mcs ef1 hygro vector

Full-length mouse Serpinb9 cDNA was amplified from the pCMV-SPORT6 vector (Dharmacon, accession BC029900, clone 4925100) using primers (forward, 5′-GCTCTAGAATGAATACTCTGTCTGAAGG-3′, reverse, 5′-CGGGATCCTGGAGATGAGAACCTGCCAC-3′) and inserted into the XbaI and BamHI sites of pCDH-CMV-MCS-EF1-Hygro vector (System Biosciences). The cloning of the vector was validated by sequencing. This vector together with pMD2.G and pCMV-dR8.74 were used for transfection of 293T cells to produce lentiviral particles. Viral particles collected from the culture supernatants were used for transfection of cancer cells using SureENTRY.

The human ARL11 expression construct (RC203868) was obtained from Origene and cloned with a C-terminal HA or Myc tag into pcDNA3.1(−) vector (Invitrogen) using XhoI and BamHI restriction sites. To construct the C-terminal GFP-tagged human Arl11, the cloning was done in the pEGFP-N1 vector (Clontech) using XhoI and BamHI restriction sites. For performing the rescue experiments, the human ARL11 gene with a C-terminal HA tag was cloned into the pCDH CMV MCS EF1-Hygro vector using NheI and BamHI restriction sites (System Biosciences). The mouse Arl11 gene with a C-terminal HA or Myc tag was cloned into pcDNA3.1(−) using cDNA prepared from RAW264.7 macrophages. To create a C-terminal TAP-tagged mouse ARL11, the cloning was performed in the pCTAP-A vector (Agilent) using BamHI and XhoI restriction sites. HA-ERK2 (MAPK1/p42)-pcDNA3 and HA-JNK1-pcDNA3 were supplied by Dr. Steve Caplan (University of Nebraska Medical Center). HA-p38-pSG5 was kindly provided by Dr. Stephen Keyse (University of Dundee). All of the point mutants were designed and obtained using Stratagene site-directed mutagenesis kits (Agilent).