Journal: PLOS One

Article Title: Minigenes for heterologous expression of human and mouse cationic trypsinogen

doi: 10.1371/journal.pone.0343840

Figure Lengend Snippet: A , Secreted trypsinogen protein (arrow) in the conditioned medium 24 hours and 48 hours after transfection was assessed by SDS-PAGE and Coomassie Blue staining. Representative gels from 3 independent transfections are shown. B , Trypsinogen levels in the conditioned medium were determined by trypsin activity measurement after activation with enteropeptidase. Individual values from 4 transfections with duplicates (n = 8) are shown, with the mean and SD indicated. C , Western blot analysis of trypsinogen (arrow) levels in cell lysates 48 hours post-transfection. Alpha-tubulin was measured as loading control. Representative blots are shown. D , PRSS1 mRNA levels were measured 48 hours after transfection by reverse-transcription quantitative PCR and expressed as fold change relative to the average value of the cDNA construct. Individual values from 3 transfections with duplicates (n = 6) are shown, with the mean and SD indicated. E , Splicing of PRSS1 mRNA expressed from cDNA and minigene constructs was analyzed 48 hours after transfection by reverse-transcription PCR and agarose gel electrophoresis. The arrow indicates the correctly spliced PRSS1 band. The smaller faint band in the minigene samples indicated by the asterisk corresponds to an aberrant splice product in which nucleotide c.40 was spliced to c.114 resulting in the deletion of the mini-intron and an extra 74 nucleotides from exon 2.

Article Snippet: The anti-PRSS1 sheep polyclonal antibody (catalog number AF3848, R&D Systems) was used at 1:5000 dilution.

Techniques: Transfection, SDS Page, Staining, Activity Assay, Activation Assay, Western Blot, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Construct, Agarose Gel Electrophoresis