Quantifying SORBS2 Isoform Expression

Total RNA was collected using RNeasy (Qiagen) from WT and SORBS2 KO MDCKII cells grown on culture dishes until confluency. Reverse transcription was performed using SuperScript^® VILO™ kit (ThermoFisher, Waltham, MA). qRT-PCR was performed as previously described [28 (link)] using primers for canine SORBS1, SORBS2, SORBS3 and ZO-1 (SORBS1 and SORBS3 primers: S2 Table, ZO-1 primers: previously published [28 (link)]).
For SORBS2 isoform identification, mRNA was collected from WT SKco15 and MDCKII cells and reverse transcription was performed as described above. Primers were designed that could identify all, or specific isoforms, of SORBS2 in canine- or human cells (SORBS2 primers, S2 Table) and DNA was amplified (Phusion, HF kit, New England Biolabs). The PCR products were loaded to a 1% agarose gel (SeaKem^®GTG^® agarose, Lonza) and separated by electrophoresis. Ethidium bromide-stained DNA bands were visualized by UV imaging (MyECL imager, ThermoFisher).