Normalization prl sv40p plasmid

For the transient reporter analyses, PD-L1 and PD-L2 promoters were amplified by PCR from genomic DNA obtained from Jurkat cells using the primers listed in Table S1 and cloned into the multicloning site (MCS) of the PGL3 Basic Vector (Promega Corporation) as previously described (Sambrook et al., 1989 ; full methods can be found in Supplemental Experimental Procedures). Specific deletions of the putative binding sites were carried out using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs), with primers and templates listed in Table S1. Normalization pRL-SV40P plasmid (Addgene plasmid 27163) was a gift from Ron Prywes (Chen and Prywes, 1999 (link)), who deposited the plasmid at the Addgene public repository.
For the stable reporter cell lines, the PD-L1Prom-DSRed-FireflyLuciferase/Neo lentiviral construct was cloned as described in Supplemental Experimental Procedures. shRNA lentiviral particles based on the pGIPZ lentiviral vector (Dharmacon) carrying hairpins for the specific genes were generated at UCLA’s Molecular Screening Shared Resource (MSSR) from the shRNA Hannon collection. Plasmids were prepared using the PureLink HiPure Filter Plasmid Maxiprep Kit (Invitrogen) or NucleoSpin 96-well Miniprep kits (Macherey-Nagel), and lentiviral particles were produced as previously described (Kappes and Wu, 2001 (link); Silva et al., 2005 (link)).