Fig 1: Characterization of Arabidopsis fpgs1, ccoaomt1, and fpgs1ccoaomt1 mutants. a Schematic diagram of the exon–intron organization of CCoAOMT1 and FPGS1 genes, and the T-DNA insertion positions in ccoaomt1 (SALK_151507) and fpgs1 mutants. b Confirmation of FPGS1 and CCoAOMT1 transcript levels in 9-day-old plate-grown WT, fpgs1, ccoaomt1and fpgs1ccoaomt1 by RT-PCR (Lane M, 1 kb DNA ladder Promega G5711; lane 1, housekeeping control AtACTIN 2 gene; lane 2, FPGS1 gene; lane 3, CCoAOMT1 gene). c 6-week-old WT, fpgs1(f1), ccoaomt1 (cc1), and fpgs1ccoaomt1 (f1cc1) Arabidopsis plants prior sampling the stems for analysis (n ≥ 30). d Comparisons of plant height, fresh stem weight, and fresh aboveground weight of 6-week-old WT, fpgs1, ccoaomt1, and fpgs1ccoaomt1 Arabidopsis plants. Plant height is the height of the primary inflorescence stem; Aboveground plant fresh weight: include all aboveground tissues including leaves, flowers, and siliques; Plant stem fresh weight: aboveground tissue after removal of rosette leaves, cauline leaves, flowers, and siliques. The data were collected from 30 plants for each genotype. There were no statistically significant differences between values according to one-way ANOVA and LSD test (values were mean ± SE. n = 30, P ≤ 0.05)
Fig 2: The combination of type and concentration of detergent in the lysis buffer and application of sonication resulted in a different yield of DNA. L: 1Kb DNA ladder (Promega #G5711) in 1% agarose gel, DNA quantification was performed using a NanoDrop 1000.
Fig 3: Epitope location and generation of DLD-1 RNF43 ΔEX8-9 clone.A. Schematic representation of RNF43 mRNA and epitope location of RNF43 antibodies. The ab217787 antibody was raised against a N-terminal epitope encoded by exons 2 and 3, while the other three antibodies were raised against epitopes encoded by exons 8 and 9. A DLD-1 cell clone was generated that entirely misses these exons leading to a p.(Glu284_Pro769delext*56) deletion on protein level. B. Confirmation of correct deletion of exons 8 and 9 on DNA level. Left panel shows PCR with primers flanking the deletion. The expected approximate 900bp fragment is observed in the ΔEX8-9 clone, while the original 4kb fragment is too big to be amplified. Middle and right panels show, respectively, PCRs for exons 8 and 9, leading to the expected 283 and 914bp fragments in the wild-type cell line, whereas only non-specific bands are observed in the ΔEX8-9 clone. DNA marker used is the 1kb DNA ladder from Promega (#G5711) C. Confirmation of correct deletion of exons 8 and 9 on mRNA level. Primers flanking exons 8 and 9 reveal the expected 1904 and 445bp fragments, respectively, for the wild-type cells and ΔEX8-9 clone. D. A quantitative RT-PCR analysis of RNF43 exons 8–9 shows undetectable levels in the ΔEX8-9 clone. Interestingly, as shown by a qRT-PCR for exons 6–7, total RNF43 levels are decreased about 200-fold in this clone. In conclusion, we have successfully generated a DLD-1 clone that shows strongly reduced levels of RNF43 mRNA entirely lacking exons 8 and 9.
Supplier Page from Promega for 1kb DNA Ladder
Fragment Sizes:250, 253, 500, 750, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 6000, 8000, 10000 bp