Figure 1.

Lentiviral-mediated production of tagged SOCS3 (SOCS3t) in rat primary glial cell cultures. Two days after transduction, the presence of SOCS3 mRNA and of the tag sequence V5 were assessed using conventional RT-PCR with 0.5 μg of total RNA extracted from primary glia transduced with LV–SOCS3t (35 ng of viral envelope protein p24) or LV–EGFP (control lentiviral vectors, 35 ng of p24) or untreated (controls). A, Socs3 and V5 mRNA levels were compared with housekeeping GAPDH mRNA levels for each condition. Western blots were performed with SOCS3- or V5-specific antibodies on total proteins extracted from glial cells, 2 d after transduction. α-Tubulin was used as a loading control. B, Addition of the V5 tag sequence allowed endogenous SOCS3 to be distinguished from transgene-derived SOCS3 protein. C, Whereas in control glia immunohistochemistry could not detect endogenous SOCS3-IR, SOCS3-IR (in red) was present in cells transfected with LV–SOCS3t (35 ng of p24, 2 d before experiments), fully overlapping with V5-immunolabeling (in green), thus confirming the transgene origin of SOCS3-IR in these cells. Scale bar, 50 μm.