Pbabe puro mcherry gfp lc3

For retroviral production, 6 μg of pBabe-puro mCherry-GFP-LC3 (Addgene #22418) or pBabe-puro-EGFP (enhanced green fluorescent protein) as a control were mixed with 0.6 μg of pV-SVGR and 5.4 μg of pGAG-POL packaging vectors (provided by Roger Gomis, IRB Barcelona) in NaCl (150 mm) supplemented with PEI (5.8 μg/ml)^{58 (link)}. HEK293T cells were plated on a 100 mm tissue culture dish, grown overnight, and transfected with the plasmid mix for further viral production at 37 °C for 48 h. The supernatant of HEK293T cells was collected and filtered through polyvinylidene difluoride filter (0.45 μm). Retroviral transduction was performed with 5 ml of media containing viral particles mixed with 5 ml of fresh media supplemented with 8 μg/ml polybrene (Sigma, 107689) in a 100 mm dish with the recipient cells for 24 h. Medium was then replaced and the cells were selected in the presence of 2 μg/ml puromycin (Sigma, P9620).

The following plasmids were from Addgene:
pMXs-puroGFP-DFCP1,pCDNA6-myc ULK1 wt, pCDNA3 Flag ULK1, pCDNA3 myc AMPK
Alpha 2, pBABE-puro-mCherry-GFP-LC3,and pBABE-puro-GFP-LC3. pCMV-AMPK alpha
2 Flag was from Sinobiologicals. Our lab generated pCMV-IPMK-GST and
different fragments tagged with GST, pCMV-myc-IPMK, pCMV-HA-IPMK, pMX-myc,
pMX-myc IPMK WT, and pMX-myc KSA. Constitutively active AMPK was a kind gift
from Dr. Anne Burnet (Stanford University School of Medicine, Stanford, CA).
We purchased recombinant hIPMK from Origene. Recombinant hULK1 and AMPK were
purchased from Signalchem.