Journal: PLoS Genetics

Article Title: A conserved, N-terminal tyrosine signal directs Ras for inhibition by Rabex-5

doi: 10.1371/journal.pgen.1008715

Figure Lengend Snippet: Table of reagents used in the manuscript with corresponding identifiers.

Article Snippet: UAS Flag-His6-Ras WT , UAS Flag-His6-Ras Y4E , UAS Flag-His6-Ras Y4F , UAS Flag-His6-Ras G12V , UAS Flag-His6-Ras Y4E,G12V , and UAS Flag-His6-Ras Y4F,G12V plasmids were sent to BestGene for injection and generation of transgenic lines at the attp40 locus.

Techniques: Cell Culture, Protease Inhibitor, Extraction, Transfection, Recombinant, Software

Journal: PLoS Genetics

Article Title: A conserved, N-terminal tyrosine signal directs Ras for inhibition by Rabex-5

doi: 10.1371/journal.pgen.1008715

Figure Lengend Snippet: Increasing temperature increases Gal4/UAS-mediated expression of transgenes. (A) Control wing ( c765-gal4/+ ) at 18°C. (B) Oncogenic Ras, Ras G12V , expressed using c765-gal4 at 18°C causes subtle vein abnormalities. (C) Y4F mutation in oncogenic Ras, Ras Y4F,G12V , shows an increase in wing vein effects. (D) Control wing ( c765-gal4/+ ) at 21°C. (D’) Control wing expressing low level of Rabex-5 DPYT using c765-gal4 at 21°C. This level of expression is not sufficient to disrupt wing vein pattern. (E) Ras G12V , expressed using c765-gal4 at 21°C, causes extra wing veins and thickened veins. (E’) Rabex-5 DPYT expression concurrent to Ras G12V , using c765-gal4 at 21°C suppresses the extra wing veins and thickened vein phenotypes. (F) Ras Y4F,G12V expressed using c765-gal4 at 21°C shows an increase in wing effects including reduction in size compared to Ras G12V . (F’) Rabex-5 DPYT expression concurrent to Ras Y4F,G12V using c765-gal4 at 21°C shows a similar phenotype as Ras Y4F,G12V . (G) Control wing ( c765-gal4/+ ) at 22°C. (H) Ras G12V , expressed using c765gal4 at 22°C causes a more severe phenotype than at 21°C. (I) Ras Y4F,G12V expressed using c765-gal4 at 22°C shows further wing disruption compared to Ras G12V . Female wings are shown; for male wings, see .

Article Snippet: UAS Flag-His6-Ras WT , UAS Flag-His6-Ras Y4E , UAS Flag-His6-Ras Y4F , UAS Flag-His6-Ras G12V , UAS Flag-His6-Ras Y4E,G12V , and UAS Flag-His6-Ras Y4F,G12V plasmids were sent to BestGene for injection and generation of transgenic lines at the attp40 locus.

Techniques: Expressing, Control, Mutagenesis, Disruption

Journal: PLoS Genetics

Article Title: A conserved, N-terminal tyrosine signal directs Ras for inhibition by Rabex-5

doi: 10.1371/journal.pgen.1008715

Figure Lengend Snippet: (A-F) Y4E phosphomimic mutation suppresses the eye overgrowth and outgrowth phenotypes of Ras G12V . Control eye ( ey-gal4/+ ) (A, left eye in D, left eye in F). Ras G12V , driven by ey-gal4 (B, right eye in D and E). Ras Y4E,G12V driven by ey-gal4 (C, left eye in E, right eye in F). Head-to-head photos in D-F highlight the suppression of overgrowth. (G) Control GMR-gal4/+ eye. (H) Ras G12V driven by GMR-gal4 . (I) Ras Y4E,G12V driven by GMR-gal4 . Y4E phosphomimic mutation suppresses the rough eye and black tissue phenotypes of Ras G12V . Female eyes are shown in A-I. For male eyes, see . (J-L) He-gal4 was used to drive Ras transgene expression in hemocytes. To visualize hemocytes, a UAS GFP transgene was also used. (J) Control, GFP driven by He-gal4 . (K). Ras G12V and GFP driven by He-gal4 . (L). Ras Y4E,G12V and GFP driven by He-gal4 . Larvae in J-L were imaged at the same settings. Tracings of larvae in J and L indicate larval outlines. Excess hemocytes are evident in (K) by the strong GFP signal (green). The excess hemocyte phenotype is suppressed upon Y4E mutation. Scale bars in J-L indicate 1.5 mm. Images of the entire larvae are shown in . (M) Control wing (c765-gal4/+) . (N) Ras G12V driven by c765-gal4 . (O) Ras Y4E,G12V driven by c765-gal4 . Y4E phosphomimic mutation suppresses the extra wing vein phenotype of Ras G12V . (P) Control homozygous MS1096-gal4 wing. (Q) Wing homozygous for MS1096-gal4 and Ras Y4E,G12V . One copy of oncogenic Ras driven by ms1096gal4 is lethal (therefore wings cannot be shown); Y4E phosphomimic mutation yields obvious wing phenotypes but suppresses the lethality of expressing two copies of Ras G12V . (R) Control c765-gal4/+ wing. (S) Wing homozygous for c765-gal4 and Ras Y4E,G12V transgene show the obvious extra wing vein phenotype associated with oncogenic Ras. (T) Low-level Rabex-5 RNAi driven by c765-gal4 yields no visible phenotype. (U) Ras Y4E,G12V expression driven by c765-gal4 shows very subtle or no extra wing vein phenotypes. (V) Ras Y4E,G12V expression elicits obvious extra wing vein phenotypes (arrows) upon concurrent low-level Rabex-5 RNAi driven by c765-gal4 . Female wings are shown in M-V; for male wings, see .

Article Snippet: UAS Flag-His6-Ras WT , UAS Flag-His6-Ras Y4E , UAS Flag-His6-Ras Y4F , UAS Flag-His6-Ras G12V , UAS Flag-His6-Ras Y4E,G12V , and UAS Flag-His6-Ras Y4F,G12V plasmids were sent to BestGene for injection and generation of transgenic lines at the attp40 locus.

Techniques: Mutagenesis, Control, Expressing

Journal: PLoS Genetics

Article Title: A conserved, N-terminal tyrosine signal directs Ras for inhibition by Rabex-5

doi: 10.1371/journal.pgen.1008715

Figure Lengend Snippet: (A) Ras WT and Ras G12V proteins incubated in the presence or absence of JAK2, EGFR, or SRC proteins. Anti-pY4 antibodies recognize a baseline level of recombinant Ras protein species (lane 1). Increased recognition of Ras WT protein by anti-pY4 antibodies is seen upon incubation with JAK2 (lane 2) and SRC (lane 4) but not EGFR (lane 3) compared to unmodified protein (lane 1). Increased recognition of Ras G12V protein by anti-pY4 antibodies is seen upon incubation with JAK2 (lane 2), EGFR (lane 3) and SRC (lane 4) compared to unmodified protein (lane 1). (B) Ras WT , Ras G12V , Ras Y4F , and Ras Y4F,G12V proteins incubated in the presence or absence of different preparations of JAK2, EGFR, or SRC proteins. Increased recognition of Ras WT and Ras G12V proteins by anti-pY4 antibodies is seen upon incubation with JAK2 (lane 5) and SRC (lane 7) but not EGFR (lane 6) compared to unmodified protein (lane 1). No difference in recognition of Ras Y4F or Ras Y4F,G12V proteins by anti-pY4 antibodies is seen upon incubation with JAK2 (lane 5), EGFR (lane 6) and SRC (lane 7) compared to unmodified protein (lane 1). Anti-pY4 antibodies also recognize JAK2 and SRC but this recognition does not interfere with detection of Ras proteins which run at a different size. (C-C’) Control GMR-gal4/+ eye shown in profile (C) and from overhead (C’). (D-D’) Ras G12V expressed using GMR-gal4 . Eyes are rough and show some loss of eye pigment. Some eyes have black tissue at the periphery of the eye (arrow) shown in profile (D) and overhead (D’). (E-E-) Ras Y4F,G12V expressed using GMR-gal4 . Some eyes have black tissue at the periphery of the eye (arrow) shown in profile (E) and overhead (E’). (F-F’) hop RNAi driven by GMR-gal4 yields no visible phenotype shown in profile (F) and overhead (F’). (G-G’) hop RNAi concurrent to Ras G12V expression using GMR-gal4 . Eyes are rough and show a more consistent appearance of black tissue (arrow, quantified in I) shown in profile (G) and overhead (G’). (H-H’) hop RNAi concurrent to Ras Y4F,G12V expression using GMR-gal4 . Eyes are rough but do not show enhancement of the black tissue (arrow, quantified in I) shown in profile (H) or overhead (H’). (I) Graph quantifying the presence of black tissue in control Ras G12V and Ras Y4F,G12V eyes or Ras G12V and Ras Y4F,G12V eyes undergoing concurrent hop RNAi. hop RNAi enhances the appearance of black tissue in Ras G12V eyes but not in Ras Y4F,G12V eyes. In the case shown, hop RNAi suppresses the appearance of black tissue in Ras Y4F,G12V eyes. Suppression was reproducible but variable; in some trials we saw no statistically significant difference between Ras Y4F,G12V eyes and Ras Y4F,G12V eyes undergoing concurrent hop RNAi. Total N is indicated below the graph, and N for each category is indicated in each section of the bar graph.*** indicates p<0.0001, and ** indicates p<0.005 from CHITEST function in Excel for Chi-square statistical analysis comparing the percentage of black tissue between the indicated genotypes. Female eyes are shown in C-H and quantified in I. Increased lethality in males in these experiments resulted in numbers too small for statistical analysis. (J) We propose a model that Ras phosphorylation at Y4 promotes ubiquitination of Ras-GDP and Ras-GTP by Rabex-5. We consistently see greater ubiquitination of Ras G12V than of Ras WT , and this is also seen for human Ras . This finding, together with additional amino acids affecting ubiquitination of Ras G12V than in Ras WT , suggest that there could be one kinase that targets both Ras-GDP and Ras-GTP and a second kinase that also targets Ras-GTP.

Article Snippet: UAS Flag-His6-Ras WT , UAS Flag-His6-Ras Y4E , UAS Flag-His6-Ras Y4F , UAS Flag-His6-Ras G12V , UAS Flag-His6-Ras Y4E,G12V , and UAS Flag-His6-Ras Y4F,G12V plasmids were sent to BestGene for injection and generation of transgenic lines at the attp40 locus.

Techniques: Incubation, Recombinant, Control, Expressing, Phospho-proteomics, Ubiquitin Proteomics

Journal: PLoS Genetics

Article Title: A conserved, N-terminal tyrosine signal directs Ras for inhibition by Rabex-5

doi: 10.1371/journal.pgen.1008715

Figure Lengend Snippet: Table of protein sequences for Ras constructs used in vitro and in vivo .

Article Snippet: UAS Flag-His6-Ras WT , UAS Flag-His6-Ras Y4E , UAS Flag-His6-Ras Y4F , UAS Flag-His6-Ras G12V , UAS Flag-His6-Ras Y4E,G12V , and UAS Flag-His6-Ras Y4F,G12V plasmids were sent to BestGene for injection and generation of transgenic lines at the attp40 locus.

Techniques: Construct, In Vitro, In Vivo, Sequencing

Journal: Science signaling

Article Title: A systems mechanism for KRAS mutant allele-specific responses to targeted therapy

doi: 10.1126/scisignal.aaw8288

Figure Lengend Snippet: (A) EGFR signals through the RAS GTPases to drive proliferation. Constitutively active Ras mutants are active in an EGFR-independent manner and are known to cause resistance to EGFR inhibitors. (B) The biochemical processes that influence Ras nucleotide binding for both wild-type and mutant Ras proteins and that are the focus of the mathematical model. (C) Simulated anti-EGFR dose response from the computational Ras model. (D) MTT proliferation assays to assess dose responses of KRAS WT SW48 (WT) colon cancer cells and three derivative isogenic cell lines, each with one of the three most common KRAS mutants in colon cancer (G12D, G12V, and G13D), to the EGFR-blocking antibody cetuximab (CTX at dose indicated for 48 hours). Data are means ± SD of seven biological replicates and are representative of three experiments. (E) Two-dimensional colony formation assay for each cell line in the isogenic panel treated without or with cetuximab (CTX; 20μg/ml) for seven days. Images are representative of six independent experiments. (F) Ras binding domain (RBD) pull-down Ras activation assays for isogenic SW48 cells cultured without and with cetuximab (as in E). Four biological replicates for each condition were included in each of three independent experiments. (G) Immunoblots of ERK phosphorylation in whole-cell lysates from isogenic SW48 cells cultured in the presence of increasing concentrations of cetuximab. Blots are representative of three independent experiments.

Article Snippet: Cell proliferation was assayed within at least 48 h. Ras expression constructs from the NCI Ras Initiative clone collection for KRAS4B-WT (Addgene #83129), NRAS-WT (Addgene #83173), HRAS-WT (Addgene #83181), KRAS-G13D (Addgene #83133), KRAS-G12V (Addgene #83132), and KRAS-G12D (Addgene #83131) were Gateway cloned into EGFP expression vector pEZYegfp (Addgene #18671).

Techniques: Binding Assay, Mutagenesis, Blocking Assay, Colony Assay, Activation Assay, Cell Culture, Western Blot

Journal: Science signaling

Article Title: A systems mechanism for KRAS mutant allele-specific responses to targeted therapy

doi: 10.1126/scisignal.aaw8288

Figure Lengend Snippet: (A) Simulated anti-EGFR dose response for the Ras model, further subdivided to reveal the change in active, GTP-bound mutant Ras (left) and the change in active, GTP-bound wild-type Ras (right), within each modeled genotype. (B) Ras binding domain (RBD) pull-down Ras activation assays for isogenic SW48 cells (WT, KRAS G12V, and KRAS G13D) cultured without or with cetuximab. CTX; 20μg/ml) or without cetuximab for 48 hours. Blots are representative of four independent experiments. (C) Densitometry-based quantification of the ratio of RasGTP between cetuximab-treated and untreated cells from three independent assays represented in (B). The quantified data are means ± SD. *P<0.05, one-way ANOVA (F=35.22) with post-hoc Tukey’s test for multiple comparisons between WT or G13D cells vs. G12V cells for each RAS isoform. (D) Mass spectrometry-based quantification of the GTP-bound wild-type HRAS, wild-type NRAS, total (both wild-type and mutant) KRAS, and wild-type H/N/KRAS in cetuximab-treated KRASG12V or KRASG13D cells relative to untreated counterparts. (CTX; 20μg/ml for 48 hours). Data from two independent experiments are presented. (E) Isoelectric focusing of excised gel bands from RBD pull-down lysates, performed upon excised gel bands. Lysates are from isogenic SW48 cells (WT, KRAS G12V, and KRAS G13D) cultured without or with cetuximab (CTX; 20μg/ml for 48 hours). Blot is representative of three independent experiments.

Article Snippet: Cell proliferation was assayed within at least 48 h. Ras expression constructs from the NCI Ras Initiative clone collection for KRAS4B-WT (Addgene #83129), NRAS-WT (Addgene #83173), HRAS-WT (Addgene #83181), KRAS-G13D (Addgene #83133), KRAS-G12V (Addgene #83132), and KRAS-G12D (Addgene #83131) were Gateway cloned into EGFP expression vector pEZYegfp (Addgene #18671).

Techniques: Mutagenesis, Binding Assay, Activation Assay, Cell Culture, Mass Spectrometry

Journal: Science signaling

Article Title: A systems mechanism for KRAS mutant allele-specific responses to targeted therapy

doi: 10.1126/scisignal.aaw8288

Figure Lengend Snippet: (A) Schematic to explain computational Ras hybrid mutants. G13D, G12D, and G12V have been described to differ in seven biochemical parameters. 648 different computational hybrids were generated by considering all of the possible combinations of these differentiating parameters. For each mutant, the model was evaluated to determine whether the computational hybrid was sensitive (“Y” in bottom row) or resistant to simulated EGFR inhibition. Orange indicates a parameter value specific to KRAS G13D, blue a parameter value specific to G12D, green a value specific to G12V, and black a value specific to KRAS WT that is also used for a mutant when do specific data is available. Fifteen representative hybrid mutants are shown from the 648 total hybrid mutants to visualize how they hybrid mutants contain combinations of the individual parameters used to model a G13D, G12D, or G12V mutant. (B) Simulated dose responses for all 648 hybrids, color coded on the basis of whether the hybrid had the Ras/NF1 Km value of the G13D mutant or that of the G12V or G12D mutant. (C) Co-immunoprecipitation of NF1 with KRAS G12V, G13D, and G12V/G13D (GG/VD) from mixtures of lysates from NF1-transfected cells with lysates from RAS-transfected cells. Blots are representative of three independent experiments. (D) MTT proliferation assays of cetuximab-treated KRAS WT SW48 cells transfected with WT, G12V, G12D, G13D, G12V/G13D double mutant (GG/VD), or both G12V and G13D KRAS. (CTX; 20μg/ml for 48 hours). Data are means ± SD of eight biological replicates and are representative of three experiments. Significance was determined with a cutoff of 25% induced growth and ****P<0.001 when compared to mock transfection by one-way ANOVA (F=90.69) with post-hoc Tukey’s test for multiple comparisons.

Article Snippet: Cell proliferation was assayed within at least 48 h. Ras expression constructs from the NCI Ras Initiative clone collection for KRAS4B-WT (Addgene #83129), NRAS-WT (Addgene #83173), HRAS-WT (Addgene #83181), KRAS-G13D (Addgene #83133), KRAS-G12V (Addgene #83132), and KRAS-G12D (Addgene #83131) were Gateway cloned into EGFP expression vector pEZYegfp (Addgene #18671).

Techniques: Generated, Mutagenesis, Inhibition, Immunoprecipitation, Transfection

Journal: Science signaling

Article Title: A systems mechanism for KRAS mutant allele-specific responses to targeted therapy

doi: 10.1126/scisignal.aaw8288

Figure Lengend Snippet: (A) In a KRAS WT cancer, NF1 ensures there are low levels of RasGTP when EGFR is not active (or is inhibited). In KRAS G12D and KRAS G12V cancers, mutant Ras is active. Wild-type Ras is also active through the competitive inhibition of NF1 through the non-productive interaction between these Ras mutants and NF1. In a KRAS G13D cancer, mutant Ras is active but wild-type Ras remains dependent on EGFR for activation due to the inability of KRAS G13D to bind NF1. (B) MTT proliferation assays for isogenic SW48 cells with siRNA knock down of NF1 and/or with cetuximab treatment. (C) MTT proliferation assays for isogenic SW48 cells with neurofibromin transfection and/or with cetuximab treatment. (D) MTT proliferation assays of cetuximab treated KRAS G12D SW48 cells (left) and KRAS G13D SW48 cells (right) transfected with KRAS WT, G12V, G12D, or G13D. For B-D, (CTX; 20μg/ml for 72 hours). Data are means ± SD of eight biological replicates and are representative of 3 experiments. *P<0.05 and **P<0.01 by one-way ANOVA (F>27 for all three graphs) with post-hoc Tukey’s test.

Article Snippet: Cell proliferation was assayed within at least 48 h. Ras expression constructs from the NCI Ras Initiative clone collection for KRAS4B-WT (Addgene #83129), NRAS-WT (Addgene #83173), HRAS-WT (Addgene #83181), KRAS-G13D (Addgene #83133), KRAS-G12V (Addgene #83132), and KRAS-G12D (Addgene #83131) were Gateway cloned into EGFP expression vector pEZYegfp (Addgene #18671).

Techniques: Mutagenesis, Inhibition, Activation Assay, Transfection

Journal: Science signaling

Article Title: A systems mechanism for KRAS mutant allele-specific responses to targeted therapy

doi: 10.1126/scisignal.aaw8288

Figure Lengend Snippet: (A) Immunoblot of NF1 expression in an extended panel of CRC cell lines that includes three KRAS G13D, NF1 mutant, CRC cell lines (LoVo, HCT116, and HCT-15), KRAS WT, NF1 WT, CaCo2 cells, KRAS G12V, NF1 WT, SW403 cells, and KRAS G12D, NF1 WT, LS180 cells. Parental, KRAS WT, NF1 WT, SW48 cells are included for comparison. Blots are representative of three independent experiments. (B) MTT proliferation assays to assess dose responses of the extended panel of CRC cell lines. (top) Dose responses for the three KRAS G13D, NF1 mutant, cell lines and for the same three cell lines that have been transduced to express NF1. (bottom) Dose responses from the three NF1 WT cell lines. (CTX at dose indicated for 48 hours). Data are means ± SD of eight biological replicates and are representative of three experiments. IC50 values are presented for sensitive cell lines; (−) indicates a resistant cell line. (C) Immunoblot of NF1 expression in the KRAS G13D, NF1 mutant, CRC cell lines (LoVo, HCT116, and HCT-15) after lentiviral transduction with NF1. Non-transduced SW48, CaCo2, SW403, and LS180 cells are included for comparison. Blots are representative of 3 independent experiments. (D) Ras binding domain (RBD) pull-down Ras activation assays and ERK phosphorylation immunoblots for CRC cell lines LoVo, HCT116, HCT-15, CaCo2, SW403, and LS180, cultured without or with cetuximab (CTX; 20μg/ml for 48 hours). The NF1 mutant cell lines were investigated both in native form and after transduction with NF1 (+NF1). Blots are representative of 3 independent experiments.

Article Snippet: Cell proliferation was assayed within at least 48 h. Ras expression constructs from the NCI Ras Initiative clone collection for KRAS4B-WT (Addgene #83129), NRAS-WT (Addgene #83173), HRAS-WT (Addgene #83181), KRAS-G13D (Addgene #83133), KRAS-G12V (Addgene #83132), and KRAS-G12D (Addgene #83131) were Gateway cloned into EGFP expression vector pEZYegfp (Addgene #18671).

Techniques: Western Blot, Expressing, Mutagenesis, Transduction, Binding Assay, Activation Assay, Cell Culture

Journal: Cell Structure and Function

Article Title: A Peptide Derived from Phosphoinositide 3-kinase Inhibits Endocytosis and Influenza Virus Infection

doi: 10.1247/csf.19001

Figure Lengend Snippet: Deletion of RAPEL in PI3K RBD resulted in the inability of the Ras-PI3K complex to undergo endosomal localization. (A, B) Multiple alignments of amino acid sequences of the RBDs of Ras effectors (A) and of 28 N-terminal amino acids of PI3K RBDs (B). “.”, “:”, and “*” indicate low homology, high homology, and identical amino acids, respectively. (C, D) Cos-1 cells were transfected with expression vectors for TagRFP-EEA-1 and VN-H-Ras together with either wild-type (WT) or RAPEL-deleted (ΔRAPEL) PI3K RBD-VC vectors. Twenty-four hours after transfection, the cells were serum-starved for 4 hours and subjected to time-lapse confocal microscopy. At time 0, the cells were exposed to 100 ng/ml EGF. Representative images at 30 min after EGF stimulation are shown (C). Bar, 10 μm. The extent of colocalization of the Ras-PI3K complexes with EEA-1 was quantified as described in the Materials and Methods and plotted over time (D). Data are presented as the mean±s.e.m. (n≥20 from three independent experiments). P <0.0001 as calculated by MANOVA. (E, F) Cos-1 cells were transfected with expression vectors for HA-tagged wild-type (WT) or RAPEL-deleted (ΔRAPEL) PI3K p110γ. After 24 hours, the cells were subjected to immunofluorescence with the use of anti-EEA-1 and anti-HA antibodies. Representative images are shown (E). Left panels are higher magnification images of the inset indicated in the most right entire-cell images. Bar, 10 μm. The extent of colocalization of PI3K p110γ with EEA-1 was quantified (F). Data are presented as the mean±s.e.m. ( n ≥40 from three independent experiments). * P <0.0001 versus WT PI3K p110γ expressing cells as calculated by Welch’s t -tests. (G, H) 293T cells were transfected with a control vector (Ctrl) or an expression vector for either WT or ΔRAPEL PI3K RBD tagged with glutathione S-transferase (GST) together with a vector for Venus-H-Ras G12V (the constitutively active form). After 24 hours, the cells were subjected to pull-down assay, followed by immunoblotting with antibodies indicated at the right. An aliquot of the cell lysate was also analyzed as a loading control. Representative immunoblots are shown (G). Band intensities were quantitated and plotted (H). Data are means±s.e.m from three independent experiments. P =0.400 between WT and ΔRAPEL as calculated by Student’s t -test.

Article Snippet: pCAGGS-VN-H-Ras WT and G12V, pCXN2-Flag-p110γ RBD-VC, pCXN2-Flag-H-Ras WT, pCAGGS-EGFP-H-Ras G12V, pCAGGS-Venus-H-Ras G12V, pCXN2-3×HA-p110γ, pCAGGS-ECFP-Rab5, and pRaichu-Ras 146X were described previously ( Fujioka et al. , 2011 ; Mochizuki et al. , 2001 ; Ohba et al. , 2003 ; Tsutsumi et al. , 2009 ). pCMV-TagRFP-EEA-1 was purchased from Addgene (Cambridge, MA, USA). cDNA for p110γ RBD ΔRAPEL was generated by PCR with the following primers: ΔRAPEL_F (5'-CCCTCGAGCAGACCATTAAGGTCTCACC-3') and RBD_R (5'-GGGCGGCCGCCGTCTGGAGGCGTGTCCAGT-3').

Techniques: Transfection, Expressing, Confocal Microscopy, Immunofluorescence, Control, Plasmid Preparation, Pull Down Assay, Western Blot

Journal: Cell Structure and Function

Article Title: A Peptide Derived from Phosphoinositide 3-kinase Inhibits Endocytosis and Influenza Virus Infection

doi: 10.1247/csf.19001

Figure Lengend Snippet: PI3P is required to endosomal localization of the Ras-PI3K complex. Cos-1 cells were transfected with expression vectors for TagRFP-EEA-1, CFP-Rab5, and VN-H-Ras G12V. Twenty-four hours after transfection, the cells were subjected to time-lapse confocal microscopy. At time 0, the cells were exposed to Vps34-IN1. Representative images are shown (A). Bar, 2 μm. The fluorescence intensities of NP within cell were quantified and plotted over time (B). Data are presented as the mean±s.e.m. ( n =12 from three independent experiments). P <0.0001 compared with before treatment, as calculated by repeated-measures ANOVA.

Article Snippet: pCAGGS-VN-H-Ras WT and G12V, pCXN2-Flag-p110γ RBD-VC, pCXN2-Flag-H-Ras WT, pCAGGS-EGFP-H-Ras G12V, pCAGGS-Venus-H-Ras G12V, pCXN2-3×HA-p110γ, pCAGGS-ECFP-Rab5, and pRaichu-Ras 146X were described previously ( Fujioka et al. , 2011 ; Mochizuki et al. , 2001 ; Ohba et al. , 2003 ; Tsutsumi et al. , 2009 ). pCMV-TagRFP-EEA-1 was purchased from Addgene (Cambridge, MA, USA). cDNA for p110γ RBD ΔRAPEL was generated by PCR with the following primers: ΔRAPEL_F (5'-CCCTCGAGCAGACCATTAAGGTCTCACC-3') and RBD_R (5'-GGGCGGCCGCCGTCTGGAGGCGTGTCCAGT-3').

Techniques: Transfection, Expressing, Confocal Microscopy, Fluorescence