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. 2017 Feb 18;36:85–92. doi: 10.1007/8904_2017_1

Fig. 1.

Fig. 1

In vitro characterization of PPT1-LAMP1 and generation of a Cre responsive transgene. (a) Expression of PPT1-LAMP1 in vitro. PPT1−/− cells were transduced with LV-Empty, LV-PPTLAMP, or LV-WTPPT. The top panel is probed for anti-hPPT1. hPPT1 has a predicted molecular weight of ~32 and 36 kDa, and hPPT1-LAMP1 is predicted at ~40 kDa. The bottom panel was probed for the C-terminus of LAMP-1. The left lanes show the markers for 30 and 40 kDa. A ~32 and ~36 kDa PPT1 signal was detected in the LV-WTPPT lane. PPT1 signal was detected at ~40 kDa in the LV-PPTLAMP lane. LAMP-1 signal was detected in the LV-PPTLAMP lane. No PPT1 or LAMP1 signal was detected in LV-Empty lane. (b) Immunofluorescence of PPT1 (green) and LAMP-2 (red). (c) Transwell experiment for “cross-correction.” Cells were transduced with LV-Empty, LV-PPTLAMP, or LV-WTPPT. There was a significant increase in enzyme activity in the LV-WTPPT cells (394.7 nmol/μg/h) compared to LV-Empty (2.7 nmol/μg/h). LV-WTPPT cells and LV-PPTLAMP cells (367.9 nmol/μg/h) had similar levels of activity. There was a significant (p < 0.05) increase in PPT1 activity in the transwell inserts (TW-cells) in the LV-WTPPT TW cells (18.5 nmol/μg/h) compared to LV-Empty TW cells (1.1 nmol/μg/h) and LV-PPTLAMP TW cells (2.2 nmol/μg/h). (d) In vitro testing of cell-specific transgene. Schematic of PPT1LAMP1 transgene is above the graph. The transgene consists of the CAGG Promoter (Chicken β-actin first exon and intron), human palmitoyl-protein thioesterase-1 (PPT1) cDNA, lysosomal-associated membrane protein (LAMP-1) cDNA, and the rabbit β-globin polyadenylation terminator (Rb β-globin Poly-(A)). A loxp-STOP-loxp (L-S-L) cassette was added to the transgene (loxp sites: black triangles). There was not a significant increase in PPT1 activity in the PPT1−/− cells transfected with the transgene alone (43.9 nmol/μg/h) or the Cre-containing plasmid alone (14.0 nmol/μg/h) compared to PPT1−/− cells (22.4 nmol/μg/h). There was an increase in PPT1 activity when the transgene and the Cre-containing plasmid were transfected together (186.4 nmol/μg/h) to WT levels (181.5 nmol/μg/h). pCAGG-hPPT1 plasmid was used as a transfection control