Fig. S2.
Adenovirus infection efficiency and additional confirmation of Cbl deletion. (A) Five-hundred thousand (5 × 105) freshly-isolated WT MECs were infected with GFP or Cre-GFP adenovirus at 4 × 108 pfu/mL and cells were observed under a fluorescence microscope the next day. Of these, 90.2% of MECs infected with GFP adenovirus and 88.7% of MECs infected with Cre-GFP adenovirus were positive for GFP. More than 250 cells for each sample were scored. (Scale bars, 20 μm.) (B, Upper) A map of the WT and mutant Cbl genomic loci. Locations of the primers used for diagnostic PCR and expected product sizes are indicated. (Lower) Genomic DNA was isolated from MECs infected with indicated adenovirus and analyzed by PCR using indicated primers. Primer F4 will not bind to genomic DNA from Cre-infected TMut MECs. In the WT allele, the expected product size between F2 and R2 is 3.5 kb, which was not amplified routinely. (C) qRT-PCR analysis of Cbl family gene transcripts after Cre adenovirus infection. Expression levels of Cbl, Cblb, and Cblc relative to Gapdh was determined in WT and TMut MECs after Cre adenovirus infection. Expression levels in WT cells are set as 1. Data represent mean ± SEM of three independent experiments.