Journal: bioRxiv

Article Title: Signaling by Ras G12V depends on EGFR activity in vivo

doi: 10.1101/2025.11.12.687982

Figure Lengend Snippet: (A) Schematic representation of the Ras1 genomic region showing the extent of the Ras1 KO deletion (“replacer region”; up) and the molecular structure of Ras1 KO-Kin constructs. From top to bottom: Ras1 WT , Flag-Ras1 WT , Flag-Ras1 G12V , Flag-KRASB WT , Flag-KRASB G12V , FRT Ras1 G12V (referred as Ras1 G12V ) and FRT Ras1 wt FRT Flag-Ras1 G12V (referred as FRT Ras1 ). (B) Representative western blot from third instar larval extracts of the following genotypes hemizygous for Ras1 : Df(3R)by10/+ (control), Flag-Ras1 wt /Df(3R)by10 , Flag-Ras1 G12V /Df(3R)by10 , Ras1 wt /Df(3R)by10 , Ras1 G12V /Df(3R)by10 and FRT Ras1/Df(3R)by10 . Note the differential migration for tagged (Flag-Ras; up) versus untagged (Ras; down) proteins. βTub was used as an internal loading control. (C) Quantification of Ras protein levels relative to βTub corresponding to three biological replicates of the western blots shown in B. Horizontal black lines indicate the mean of biological replicates. Each vertical blue line indicates the standard deviation (SD) of technical replicates, and the circles represents the mean of each biological replicate. (D) Percentage of pupal lethality in control and Ras1 KO-Kin heterozygous backgrounds. The bar graphs display the mean ± SD. (E) Survival curves of heterozygous females for control, Ras1 WT /+ (blue line) , Ras1 G12V /+ (dotted blue line) , Flag-Ras1 WT (pink line) , Flag-Ras1 G12V /+ (dotted pink line) , Flag-KRASB WT /+ (purple line) , and Flag-KRASB G12V /+ (dotted purple line) heterozygous females. Survival statistic were calculated using Gehan-Breslow-Wilcoxon test. (F) Adult female wing phenotypes of heterozygous Ras1 WT and KRASB WT (left), heterozygous Ras1 G12V and KRASB G12V (“Endogenous Ras G12V expression”), sal EPv -Gal4 UAS-GFP/UAS-Ras1 G12V (sal>UAS-Ras1 G12V ) and sal EPv -Gal4 UAS-GFP/UAS-KRASB G12V (sal>UAS-KRASB G12V ; “Ectopic Ras G12V expression”), and functional rescue of Ras1 knockdown ( sal EPv -Gal4 UAS-GFP/UAS-Ras1-RNAi ; sal>UAS-Ras1-i) by endogenous KRASB WT expression in sal EPv -Gal4 UAS-GFP/UAS-Ras1-RNAi; KRASB WT /+ flies (sal>UAS-Ras1-i; KRASB WT /+; “KRASB functional equivalence”).

Article Snippet: Briefly, Drosophila Ras1 genomic DNA from vasa-Cas9 flies, Ras1 cDNA (RE53955, DGCR) and human KRASB cDNA (SC109374, OriGene Technologies) were cloned into reintegration vectors ( RIV white and RIV FRTMCSFRTMCS3 ) ( Baena-Lopez et al , 2013 ) in either wild-type or G12V mutant forms and tagged or not with the Flag or HA epitopes at their 5’ end.

Techniques: Construct, Western Blot, Control, Migration, Standard Deviation, Expressing, Functional Assay, Knockdown

Journal: bioRxiv

Article Title: Signaling by Ras G12V depends on EGFR activity in vivo

doi: 10.1101/2025.11.12.687982

Figure Lengend Snippet: (A) Representative western blots of Raf-RAS binding domain (RBD) pulldowns detecting Ras-GTP from third instar larval extracts of the following genotypes: controls, Ras1 WT /+, Ras1 G12V /+, Flag-Ras1 WT /+, Flag-Ras1 G12V /+, Flag-KRASB WT /+, Flag-KRASB G12V /+ and FRT Ras1/+ (left) and control, Flag-Ras1 WT /+, Flag-Ras1 G12V /+ and Flag-Ras1 G12V-CAAX /+ (right). Each line shows the presence of Flag-Ras and Ras in the pulldown (above) and input (below). (B) Wing of Flag-Ras1 G12V-CAAX /+ genotype showing normal wing size and pattern of veins. (C) Quantification of Ras-GTP (% Ras-GTP) corresponding to the western blots shown in A. The graph displays the mean ± SD of different biological replicates (dots). (D) Representative western blot from third instar larval extracts from hemizygous control ( Df(3R)by10/+ ), Ras1 WT /Df(3R)by10, Ras1 G12V /Df(3R)by10, Flag-Ras1 WT /Df(3R)by10, Flag-Ras1 G12V /Df(3R)by10, Flag-KRASB WT /Df(3R)by10 and Flag-KRASB G12V /Df(3R)by10 . Blots were probed for dpERK, total ERK and βTub (loading control). (E) Quantification of dpERK levels relative to βTub corresponding to the western blots shown in D. No significant differences were observed between genotypes. Horizontal black lines indicate the mean of biological replicates. Each vertical blue line indicates the standard deviation (SD) of technical replicates, and the circles represents the mean of each biological replicate. (F) Schematic representation of a mature third instar larval wing disc indicating the pattern of dpERK accumulation by shades of red. In the wing pouch region, maximal accumulation of dpERK is detected in the developing wing veins (L2-L5), along two stripes abutting the dorso-ventral boundary (D/V) and in the precursor cells of the sensory organs (SOPs) in the dorsal notum region. (G) Immunostaining for dpERK in third instar larval wing imaginal discs of the following genotypes: Ras1 WT /+ , Ras1 G12V /+ , KRASB WT /+ and KRASB G12V /+ (top row from left to right) and Ras1 WT /Df(3R)by10 , Ras1 G12V /Df(3R)by10 , KRASB WT /Df(3R)by10 and KRASB G12V /Df(3R)by10 (bottom row, left to right). Scale bar: 100 μm. (H) Quantification of the ratio of dpERK levels between the vein L4 and the L4-L5 intervein territories in Ras1 WT /+ and Ras1 G12V /+ dorsal and ventral wing disc compartments. Violin plots display the median and the Q1 and Q3 interquartile ranges.

Article Snippet: Briefly, Drosophila Ras1 genomic DNA from vasa-Cas9 flies, Ras1 cDNA (RE53955, DGCR) and human KRASB cDNA (SC109374, OriGene Technologies) were cloned into reintegration vectors ( RIV white and RIV FRTMCSFRTMCS3 ) ( Baena-Lopez et al , 2013 ) in either wild-type or G12V mutant forms and tagged or not with the Flag or HA epitopes at their 5’ end.

Techniques: Western Blot, Binding Assay, Control, Standard Deviation, Immunostaining

Journal: bioRxiv

Article Title: Signaling by Ras G12V depends on EGFR activity in vivo

doi: 10.1101/2025.11.12.687982

Figure Lengend Snippet: (A) Accumulation of dpERK in third instar wing imaginal discs, from younger (A 1 ) to older (A 4 ). (B) Schematic representation of a mature wing disc of ap-Gal4 UAS-GFP/UAS-FLP; FRT Ras1 WT FRT Ras1 G12V /+ genotype showing the generation of dorsal Ras1 G12V /+ (GFP positive) and ventral Ras1 WT /+ (GFP negative) territories. (C) Expression of GFP (green in C 1 -C 3 ) and dpERK (red in C 1 -C 3 and white in C 1 ’-C 3 ’) in progressively older third instar wing discs of ap-Gal4 UAS-GFP/UAS-FLP; FRT Ras1 WT FRT Ras1 G12V /+ genotype. (D) Quantification of dpERK levels in dorsal (Ras1 G12V /+) relative to ventral (Ras1 WT /+) compartments in the L4-L5 intervein region of early and late third instar wing disc. Violin plots display the median and the Q1 and Q3 interquartile ranges. (E) Schematic representation of a mature wing disc of ap-Gal4 UAS-GFP/UAS-FLP; FRT HA-Ras1 WT FRT Flag-KRASB G12V /+ genotype showing dorsal KRASB G12V /+ (GFP positive) and ventral Ras1 WT /+ (GFP negative) territories. (F) Expression of GFP (green in F 1 -F 3 ) and dpERK (red in F 1 -F 3 and white in F 1 ’-F 3 ’) in progressively older third instar wing discs of ap-Gal4 UAS-GFP/UAS-FLP; FRT HA-Ras1 WT FRT Flag-KRASB G12V /+ genotype. (G) Quantification of dpERK levels in dorsal (KRASB G12V /+) relative to ventral (Ras1 WT /+) compartments in the L4-L5 intervein region of early and late third instar wing disc. Violin plots display the median and the Q1 and Q3 interquartile ranges. Scale bar: 100 μm.

Article Snippet: Briefly, Drosophila Ras1 genomic DNA from vasa-Cas9 flies, Ras1 cDNA (RE53955, DGCR) and human KRASB cDNA (SC109374, OriGene Technologies) were cloned into reintegration vectors ( RIV white and RIV FRTMCSFRTMCS3 ) ( Baena-Lopez et al , 2013 ) in either wild-type or G12V mutant forms and tagged or not with the Flag or HA epitopes at their 5’ end.

Techniques: Expressing