Journal: Journal of Biological Chemistry

Article Title: Human Intestinal Epithelial Cell Survival and Anoikis

doi: 10.1074/jbc.m405323200

Figure Lengend Snippet: FIG. 1. Expression of Akt isoforms, SI, and Fak during human enterocytic differentiation. A, representative RT-PCR analyses of the expression of Akt-1 and Akt-2 mRNA in Caco-2/15 cells at 2 (subconfluent, undifferentiated; lane 1), 0 (confluent; lane 2), 5 (lane 3), 10 (lane 4), 20 (lane 5) ,and 30 (differentiated; lane 6) PC. Total RNA was extracted and reversed-transcribed, and then 20 cycles of amplification were performed using primers specific for human Akt-1/PKB and Akt-2/PKB. S14 mRNA expression was likewise analyzed for normalization purposes. L, 100-bp DNA size markers. B, same as in A except that the mRNA expression of SI, an enterocytic differentiation marker, was also analyzed. Furthermore, amplified bands were semi-quantified, and the relative levels of Akt-1 (gray columns), Akt-2 (open columns), and SI (filled columns) were determined as ratios relative to S14. C, representative WB analyses of the expression of p57Akt-1, p57Akt-2, and p125Fak in Caco-2/15 cells at the same time points as in A. Total proteins (50 g/well) were separated by SDS-PAGE under reducing conditions, electrotransferred onto nitrocellulose membranes, and then probed with specific antibodies for the detection of Akt-1, Akt-2, and Fak. Detection of K18 was likewise analyzed for normalization purposes. D, same as in C, except that the expression of p220SI was also analyzed. Furthermore, amplified bands were semi-quantified, and the relative levels of Akt-1 (gray columns), Akt-2 (open columns), Fak (filled columns), and SI (dark-gray columns) were determined as ratios relative to K18. B and D, statistically significant (0.001 p 0.01) differences between 2 (Undifferentiated) and 30 (Differentiated) PC Caco-2/15 cells are indicated by *.

Article Snippet: Materials—The antibodies used are as follows: mAb4.47 (Upstate Biotechnology, Inc., Lake Placid, NY) directed against p125Fak; Ab07012 (Upstate Biotechnology, Inc.) directed to the phosphotyrosine 397- activated form of p125Fak (ptyr397p125Fak); Ab06-558 (Upstate Biotechnology, Inc.), directed to p57Akt-1; Ab9271 (Cell Signaling Technology, Beverly, MA), directed to the phosphoserine 473-activated form of p57Akt-1 (pser473p57Akt-1); Ab07-372 (Upstate Biotechnology, Inc.) and Ab124002 (Calbiochem), both directed against p57Akt-2; AbpS (Research Diagnostics, Flanders, NJ), directed to phosphoserine residues; mAbM5 (Sigma), directed against the FLAG epitope tag sequence; Ab7392 (Santa Cruz Biotechnology, Santa Cruz, CA), directed to the HA epitope tag sequence; mAb HSI-14, directed to sucrase-isomaltase (p220SI; kindly provided by A. Quaroni, Cornell University, Ithaca, NY); Abs 9661 and 9492, directed against CASP-3 and CASP-7, respectively (New England Biolabs, Mississauga, Ontario, Canada); and mAbCY-90 (Sigma), directed to human K18.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Marker, SDS Page

Journal: Journal of Biological Chemistry

Article Title: Human Intestinal Epithelial Cell Survival and Anoikis

doi: 10.1074/jbc.m405323200

Figure Lengend Snippet: FIG. 4. Activation of Fak within the context of human entero- cytic apoptosis/anoikis. A, representative WB analyses of Fak acti- vation from undifferentiated (2 PC; U; lanes 1, 3, 5, and 7) and differentiated (30 PC; D; lanes 2, 4, 6, and 8) Caco-2/15 cells that were maintained for 24 h in serum-free medium (Control; lanes 1 and 2) with the addition of 1 M cytochalasin D (lanes 3 and 4), 30 M Ly294002 (lanes 5 and 6), or kept in suspension (Susp.; lanes 7 and 8). Cells were lysed, and Fak (IP: Fak) was immunoprecipitated with specific antibod- ies. Total proteins from immunoprecipitates (50 g/well) were sepa- rated by SDS-PAGE under reducing conditions, electrotransferred onto nitrocellulose membranes, and probed with specific antibodies for the detection of the activated phosphorylated form of Fak (WB: ptyr397p125Fak). Membranes were thereafter reprobed with the antibod- ies used for IP in order to detect the Fak (WB: p125Fak) protein. B, same as in A except that cells were also exposed to 100 g/ml nonimmune mouse IgGs or 50 g/ml of the 1 integrin-blocking mAb P4C10 (P4C10). Immunoreactive bands were then semi-quantified, and the relative activated levels of Fak were established, which in turn were compared with those of control cultures, 100 (expressed as % of control). Statis- tically significant (0.001 p 0.01) differences between apoptosis/ anoikis-inducing treatments and control cultures are indicated by *.

Article Snippet: Materials—The antibodies used are as follows: mAb4.47 (Upstate Biotechnology, Inc., Lake Placid, NY) directed against p125Fak; Ab07012 (Upstate Biotechnology, Inc.) directed to the phosphotyrosine 397- activated form of p125Fak (ptyr397p125Fak); Ab06-558 (Upstate Biotechnology, Inc.), directed to p57Akt-1; Ab9271 (Cell Signaling Technology, Beverly, MA), directed to the phosphoserine 473-activated form of p57Akt-1 (pser473p57Akt-1); Ab07-372 (Upstate Biotechnology, Inc.) and Ab124002 (Calbiochem), both directed against p57Akt-2; AbpS (Research Diagnostics, Flanders, NJ), directed to phosphoserine residues; mAbM5 (Sigma), directed against the FLAG epitope tag sequence; Ab7392 (Santa Cruz Biotechnology, Santa Cruz, CA), directed to the HA epitope tag sequence; mAb HSI-14, directed to sucrase-isomaltase (p220SI; kindly provided by A. Quaroni, Cornell University, Ithaca, NY); Abs 9661 and 9492, directed against CASP-3 and CASP-7, respectively (New England Biolabs, Mississauga, Ontario, Canada); and mAbCY-90 (Sigma), directed to human K18.

Techniques: Activation Assay, Control, Suspension, Immunoprecipitation, SDS Page, Blocking Assay

Journal: Leukemia

Article Title: Internal tandem duplication of the FLT3 gene is a novel modality of elongation mutation which causes constitutive activation of the product.

doi: 10.1038/sj.leu.2401130

Figure Lengend Snippet: Figure 1 (a) Amino acid sequences duplicated by FLT3/ITD (Mt1 to Mt5) or inserted (Mt6). Germ line amino acid sequence of the JM is presented at the top. The TK domain starts at 608E. Non-coded amino acids sequences were added between the duplication due to various mechanisms as described previously. (b) Tyrosine-phosphorylation of FLT3 expressed in Cos 7 cells. The wild or the mutant type of FLT3 cDNA was transfected into Cos 7 cells. The lysates were immunopreci- pitated by the anti-FLT3 antibody and PA-agarose. The precipitated samples were detected by immunoblot analysis using anti-FLT3 anti- body or anti-phosphotyrosine antibody. IP, immunoprecipitation; IB, immunoblot. (c) Constitutive tyrosine-phosphorylation of the wild FLT3 by the co-transfection of the mutant FLT3. The wild and the mutant type of FLT3 were simultaneously transfected into Cos 7 cells.

Article Snippet: The membrane was incubated in mouse anti-phosphotyrosine antibody for 1 h, then in HRPlabeled goat anti-mouse IgG antibody (BioRad) for 1 h. Signals were developed as described above.

Techniques: Sequencing, Phospho-proteomics, Mutagenesis, Transfection, Western Blot, Immunoprecipitation, Cotransfection

Journal: Leukemia

Article Title: Internal tandem duplication of the FLT3 gene is a novel modality of elongation mutation which causes constitutive activation of the product.

doi: 10.1038/sj.leu.2401130

Figure Lengend Snippet: Figure 2 Cross-linking experiment. The wild or mutant FLT3 was cross-linked by BS3 and detected by immunoblot analysis using anti- phosphotyrosine antibody.

Article Snippet: The membrane was incubated in mouse anti-phosphotyrosine antibody for 1 h, then in HRPlabeled goat anti-mouse IgG antibody (BioRad) for 1 h. Signals were developed as described above.

Techniques: Mutagenesis, Western Blot

Journal: Leukemia

Article Title: Internal tandem duplication of the FLT3 gene is a novel modality of elongation mutation which causes constitutive activation of the product.

doi: 10.1038/sj.leu.2401130

Figure Lengend Snippet: Figure 3 The role of Y589 and Y591 in wild and mutant types of FLT3. The Y589 and Y591 were converted to phenylalanine. The precipitated samples by the anti-FLT3 antibody were detected by immunoblot analysis using anti-phosphotyrosine antibody. The ligand-dependent phosphorylation was decreased in a Y589F substi- tution of wild FLT3 (a). A series of Y589F substitutions of mutant FLT3 showed constitutive phosphorylation. Similarly a series of Y591 substitutions did not affect phosphorylation (b).

Article Snippet: The membrane was incubated in mouse anti-phosphotyrosine antibody for 1 h, then in HRPlabeled goat anti-mouse IgG antibody (BioRad) for 1 h. Signals were developed as described above.

Techniques: Mutagenesis, Western Blot, Phospho-proteomics