X tremegene hp

Protein A beads (KANEKA KanCapA^™) were from Wako Chemicals (Osaka, Japan). X-tremeGENE HP was purchased from Sigma (St Louis, MO, USA). Sepasol was purchased from Nacalai Tesque (Kyoto, Japan). The anti-podoplanin antibody (NZ-1, rat IgG) was purchased from Angio Bio Co. (Del Mar, CA, USA) and the anti-CLEC-2 antibody (AF1718, goat IgG) was from Novus Biologicals (Littleton, CO, USA). Horseradish peroxidase (HRP)-labeled anti-human IgG Fab2 antibody (709–1317) was purchased from Rockland Immunochemicals Inc. (Limerick, PA, USA), anti-goat IgG-HRP (P0160) from Dako (Glostrup, Denmark) and anti-Rat IgG-HRP (7077S) from Cell Signaling Tech (Danvers, MA, USA). A set of plasmids for constructing a lentivirus expression vector system, including human immunodeficiency virus type 1 (HIV-1)-based CSII-MCS-Venus, pCAG-HIVgp, and pCMV-VSV-G-RSV-Rev, were obtained from RIKEN (Wako, Osaka, Japan). The Fc-fusion protein expression plasmid containing human IgG1 (pFUSEN-hG1Fc) was purchased from InVIVOGen (San Diego, CA, USA). Hematoporphyrin was from MedChemExpress (Monmouth Junction, NJ, USA). 2CP was synthesized as reported previously [10 (link)].

PAPS synthase protein variants with preferred nuclear or cytoplasmic localization have been described previously (18 (link)). Mutating K9A,K10A in PAPSS1 or K6A,K8A in PAPSS2 disrupts a conserved nuclear localization signal and results in preferential cytoplasmic localization. Changing R111A,R112A in PAPSS1 or R101A,R102A in PAPSS2, on the other hand, inactivates a motif with nuclear export signal activity, resulting in pronounced nuclear accumulation (18 (link)). Coding sequences for all these protein variants without stop codons were NheI/BamHI inserted in the eukaryotic expression vector pEGFP-N1 with a C-terminal EGFP fusion. HEK293 cells were transiently co-transfected with these plasmids and a SULT2A1 expression vector (14 (link)) using XtremeGene HP (Sigma) according to the manufacturer's protocol.

mCherry-Lifeact was purchased from Addgene (#54491; Cambridge, MA), and pEGFP-Myo10 was previously described^{5 (link)}. To knockdown mouse MYO10 expression, we used the pLKO.1-TRC lentiviral vector (MISSION shRNA, Sigma-Aldrich) containing shRNA against mouse MYO10 (shRNA-1;TRCN0000110606, shRNA-2;TRCN0000110608, and shRNA-3;TRCN0000379126). A non-target shRNA vector (SHC016; Sigma-Aldrich) was used as a control for the shRNA experiments. For lentiviral packaging, psPAX2 and pMD2.G vectors (Addgene plasmids #12260, #12259) were co-transfected with the pLKO.1 into HEK293T cells with X-tremeGENE HP (Sigma-Aldrich) according to the manufacturer’s instructions. Virus-containing medium was harvested 72 hrs after transfection and filtered with 0.45 μm filters (EMD Millipore, Billerica, MA). Melb-a or B16F1 cells were infected with culture supernatants from HEK293T cells at a 1:6 dilution in complete culture medium. Cells were passed after 24 hrs and selected with 4 μg/ml puromycin for 14 days.