#Prepros unhelpful error message pro#
Subsequently, Western blotting analysis showed that GFP was significantly degraded to ~40% of the control group with the appearance of GFP PrePro (Fig 1D), which confirmed that PREDATOR Pro could be used to degrade target protein with high efficiency. Similar to previous ratio screening results, ~55% decrease of green fluorescence was observed in pEGFP plasmid and GFP PrePro plasmid co-transfected groups 48 hours after transfection comparing to the pEGFP plasmid and control plasmid co-transfected group(Fig 1C). Under such transfection ratio, further experiments were conducted in HEK-293T cells to characterize the performance of the GFP PrePro system. (Click here to know more about model)With their advice, the plasmid ratio was set as 1:1 in further experiment. Furthermore, we provided the data to Model hoping that they could obtain the optimal concentration ratio of GFP and GFP PrePro plasmids. Through the difference of color shade (the lighter the color, the better the degradation effect), a GFP PrePro dose dependent GFP degradation could be clearly observed. Fluorescent intensity (Click here for more about model results)was then transformed into heatmap for better visualization. To find the optimal GFP PrePro transfection dosage, gradient amount of GFP-expression plasmid, and GFP PREDATOR Pro plasmid were co-transfected into HEK-293T cells (Fig 1B, left panel), fluorescent images were taken 48 hours post transfection. Statistical significance was calculated via either student t test or two way ANOVA test. Error bars represent at least three biological replicates. Fluorescent intensity of each well were calculated by averaging the fluorescent intensity in at least three random visions. (F) Representative fluorescence images in (E). (E) Quantified fluorescent intensity in HEK-293T cell imaged 12-144 h post co-transfection with pEGFP plasmid and GFP PrePro /control plasmid (Dots with error bar), the modeling predictions were shown in curve. Relative protein level was calculated by normalizing the gray scale data of each group to the control group. (D) Representative Western blotting determining the GFP plasmid protein abundance in HEK-293T cells 48 h post co-transfected with pEGFP plasmid and GFP PrePro/control plasmid (left panel), and quantified GFP protein level in three biological replicates (right panel).
(C) Fluorescence images and quantified fluorescent intensity of HEK-293T cells 48 h post co-transfection with pEGFP plasmid and GFP PrePro/control plasmid.
GFP PREDATOR Pro system based on the DocS-Coh2 interaction module dramatically decrease cellular GFP level throughout a long time span.(A) Schematic representation showing the design and function of GFP PrePro (B) Schematic representation showing the experimental workflow (left panel) and quantified GFP fluorescence heatmap 48 h post transfection of corresponding amount of plasmid (right panel).