Cold fusion cloning kit

Nbs were cloned into mitochondrial outer membrane (MOM) V5 pcDNA3.1 His₆ and pEGFP-N1 vectors (Clontech, Mountain View, CA, USA), as described before [26 (link)–28 (link)]. The nanobodies were subcloned by means of a Cold Fusion Cloning Kit (System Biosciences, Mountain View, CA, USA). The first subcloning was performed into V5 pcDNA3.1 His₆ vector by using the following primers: forward 5’-TCGATTCTACGCGTACCGGTGCCCAGGTGCAGCTGCAGGAG-3’ and reverse 5’-GGTGATGATGACCGGCTAGCTGGAGACGGTGACCTG-3’. Cloning of the Nbs in the pEGFP-N1 vector was done using the following primers: forward 5’-CGAGCTCAAGCTTCGGCCACCATGCAGGTGCAGCTGCAGGAG-3’ and reverse 5’-GGCGACCGGTGGATCCTTGCTGGAGACGGTGACCTG-3’.
To obtain inducible Nb expressing cells, a third cloning of the EGFP-tagged VCA Nbs into the pLVX-TP vector (Clontech) was done using following primers: Forward 5’-TGGAGAAGGATCCGCGGCCGCGCCACCATGGCCCAGGTGCAGCTGCAGGAGTCTGGG-3’ and reverse 5’-CTACCCGGTAGAATTCTTACTTGTACAGCTCGTCCATGCC-3’.

Plasmids containing constructs coding for HA-AKT1, Myc-AKT1-E17K, HA-AKT2 and HA-AKT3 were kind gifts from Donghwa Kim (H. Lee Moffit Cancer Center and Research Institute). cDNA encoding the PH-domains were isolated by PRC amplification using the following primers: Akt1PH Forward (Fwd) 5’ AGC GAA TTC ATG AGC GAC GTG GCT ATT GTG 3’, Akt1PH Reverse (Rev) 5’ GAA GCT TTC AGT TGT CAC TGG GTG AGC CCG ACC G 3’, Akt2PH Fwd AGC GAA TTC ATG AAT GAG GTG TCT GTC ATC 3’, Akt2PH Rev 5’ GAA GCT TTC ACA TCC ACT CCT CCC TCT CGT CTG G 3’, Akt3PH Fwd 5’ AGC GAA TTC ATG AGC GAT GTT ACC ATT GTG 3’ and Akt3PH Rev 5’ GAA GCT TTC AAT TCA TTC TCT CCT CTT CTT GCC TCT GC 3’. Constructs were inserted into a pHEN6 vector backbone using the Cold Fusion™ cloning kit (System Biosciences) according to the manufacturers’ protocol and proteins were expressed in BL21 E. coli.

Total RNA and complementary DNA (cDNA) were obtained from E13.5 lungs using TRIzol reagent (Life Technologies) and Superscript III reverse transcriptase (Life Technologies), respectively, according to manufacturer’s instructions. cDNA encoding the MYH10 (NCBI Acc. No. NM_175260) wild-type and MYH10^L458R proteins was PCR amplified using total cDNA from E13.5 lungs as a template. PCR fragments were cloned into the pcDNA3.1-Myc-His vector at the HindIII site using the Cold Fusion Cloning Kit (System Biosciences). Thbs2 (NCBI Acc. No. NM_011581) cDNA was PCR amplified using total cDNA from E18.5 lungs as a template. PCR fragments were ligated to the pcDNA3.1-Myc-His vector at XhoI/EcoRI sites. The following primers were used: Myh10 forward 5ʹ-TCCGAGCTCGGTACCACTGTTTACAATGGCCCAGAG-3ʹ and Myh10 reverse 5ʹ-TTGTTCGGGCCCAAGCTCTGATTGGGGTGGCTGTG-3ʹ; Thbs2 forward 5ʹ-AGGTCGACGTCACAGGTGGAGACAAGATG-3ʹ and Thbs2 reverse 5ʹ-TGGAATTCTCGAGGCATCTCTGCACTCA-3ʹ. To synthesize RNA probes for in situ hybridization, cDNA was PCR amplified using total cDNA from E13.5 lungs as a template. PCR fragments were cloned into the pGEM T-easy vector (Promega). The following primers were used: Myh10 forward 5ʹ-GTACAGAAAGCCCAGACCAAAGA-3ʹ and Myh10 reverse 5ʹ-TTCTTCATCAGCCACTCATCTGC-3ʹ; Tbx4 forward 5ʹ-CTTCTACCACTGCCTGAAGCGT-3ʹ and Tbx4 reverse 5ʹ-AGTCTCGTCATCCATCGGTCCA-3ʹ.

Anti-FAK[pY397] was from BD Biosciences. Anti-tubulin antibody was from Sigma. DyLight 549 conjugated goat anti-mouse IgG (H + L) was from Thermo Scientific. Fibronectin and recombinant human EGF were from Akron Biotech; Growth factor reduced Matrigel was from BD Bioscience. Pfu Ultra was from Agilent Technologies. Cold Fusion Cloning Kit was from System Biosciences (Palo Alto, CA). Anti-GFP monoclonal antibody and Safectine RU50 transfection kit were purchased from Syd Labs (Malden, MA). DNA primers were synthesized by Sigma-Aldria.

For making retrovirus, mCherry-Borealin^WT or mCherry-Borealin^MTBM transgenes were cloned into pBABE-Puro retrovirus vector using cold fusion cloning kit (System Biosciences). HEK-293GP cells were co-transfected with pBABE-Puro-mCherry-Borealin (WT or MTBM) and VSVG plasmid in order to package pseudotyped MULV viruses. The viruses were collected 3 days post transfection by filtering the media through 0.45 μm syringe filter.