polyclonal igg antibodies against palladin  (Proteintech)

Journal: International journal of oncology

Article Title: Palladin, an actin-associated protein, is required for adherens junction formation and intercellular adhesion in HCT116 colorectal cancer cells.

doi: 10.3892/ijo_00000742

Figure Lengend Snippet: Figure 4. Effects of palladin knockdown on intercellular adhesion, collective migration and localization. A, Western blot analysis of palladin expression in HCT116 and E1 cells, HCT116 cells transfected with control RNAi (GCctl), RNAi #1, #2, #3 or #4. RNAi #1 and #4 were designed to knockdown only the 140 kDa palladin isoform, RNAi #2 and #3 were designed to knockdown both the 90 and 140 kDa palladin isoforms. GAPDH was used as a loading control. HCT116 and E1 cells were cultured to a similar density (~80%) before protein extraction. Transfected cells were harvested for protein extraction at 48 h post- transfection. Similar profiles were obtained with cells harvested at 24, 72 and 96 h post-transfection (data not shown). B, Suspension cultures of HCT116 and E1 cells, HCT116 cells transfected with control RNAi (GCctl), RNAi #1, #2, #3 or #4. RNAi-transfected HCT116 cells were re-seeded into the low cluster plates at 24 h post-transfection. Cells were cultured in suspension for 24 h in the tissue culture incubator, after which images were taken with a camera attached to the Zeiss Axiovert microscope equipped with a x10 phase-contrast objective. C, Still images of the wound fronts of untransfected HCT116, E1 cells and HCT116 cells transfected with control RNAi (GCctl), RNAi #1, #2 or #3. Transfected cells were re-seeded for the wound healing assay at 24 h post- transfection. Cells were seeded at the appropriate density such that they were 100% confluent after 24 h culture, upon which the monolayer was artificially wounded by scratching with a pipette tip. These images were captured at 24 h post-wounding.

Article Snippet: Polyclonal antibodies against palladin were purchased from Proteintech Group Inc., while those against total p44/42 MAP kinase (Erk1 and Erk2) and phospho-44/42 MAP kinase (Erk1 and Erk2) from Cell Signaling Technology.

Techniques: Knockdown, Migration, Western Blot, Expressing, Transfection, Control, Cell Culture, Protein Extraction, Suspension, Microscopy, Wound Healing Assay, Transferring

Journal: International journal of oncology

Article Title: Palladin, an actin-associated protein, is required for adherens junction formation and intercellular adhesion in HCT116 colorectal cancer cells.

doi: 10.3892/ijo_00000742

Figure Lengend Snippet: Figure 5. Effects of palladin knockdown on the expression and localization of palladin. A, Confocal microscopy images of HCT116 and E1 cells fluorescently stained with anti-palladin antibody. Two focal planes were captured for HCT116 cells: 1, plane 1 shows palladin staining at the focal adhesions, spike-like filopodia structures and 2, plane 2 shows the staining at adherens junction. B, Confocal microscopy images of HCT116 cells constained with anti-palladin antibody and either anti-E-cadherin antibody (upper panel) or anti-·-actinin antibody (lower panel). C, Immunoprecipitates from the cell lysate using anti- palladin antibody were probed with anti-·-actinin antibody, which indicated that ·-actinin co-precipitated with palladin in HCT116 cells (upper panel). Samples of whole cell lysate were run as input control (lower panel). E1 sample that contained minimal palladin was run as a negative control. D, Confocal microscopy images of HCT116 cells transfected with control RNAi (GCctl) or RNAi #2, co-stained with anti-palladin and anti-E-cadherin antibodies. Staining was carried out on transfected cells at 24 h post-transfection.

Article Snippet: Polyclonal antibodies against palladin were purchased from Proteintech Group Inc., while those against total p44/42 MAP kinase (Erk1 and Erk2) and phospho-44/42 MAP kinase (Erk1 and Erk2) from Cell Signaling Technology.

Techniques: Knockdown, Expressing, Confocal Microscopy, Staining, Control, Negative Control, Transfection

Journal: International journal of oncology

Article Title: Palladin, an actin-associated protein, is required for adherens junction formation and intercellular adhesion in HCT116 colorectal cancer cells.

doi: 10.3892/ijo_00000742

Figure Lengend Snippet: Figure 5. Continued. E, Protein extracts of HCT116, E1 cells and HCT116 cells transfected with control RNAi (GCctl) or RNAi #2 (at 24 h post- transfection) were probed for palladin and E-cadherin. There was an almost complete knockdown in palladin expression in HCT RNAi #2 cells, without any changes in E-cadherin expression.

Article Snippet: Polyclonal antibodies against palladin were purchased from Proteintech Group Inc., while those against total p44/42 MAP kinase (Erk1 and Erk2) and phospho-44/42 MAP kinase (Erk1 and Erk2) from Cell Signaling Technology.

Techniques: Transfection, Control, Knockdown, Expressing

Journal: International journal of oncology

Article Title: Palladin, an actin-associated protein, is required for adherens junction formation and intercellular adhesion in HCT116 colorectal cancer cells.

doi: 10.3892/ijo_00000742

Figure Lengend Snippet: Figure 6. Effects of the inhibition of the Mek/Erk pathway on the morphology of E1 cells and the expression and localization of palladin. A, Phase-contrast microscopy showing the morphology of: HCT116 cells, untreated E1 cells, or E1 cells treated with DMSO (control), 10, 25 or 50 μM of U0126. B, Suspension cultures of: E1 cells treated with DMSO or 25 μM of U0126 (top panel); E1 cells transfected with control RNAi (middle panel) or RNAi #2 (bottom panel), followed by treatment with DMSO (left column) or 25 μM of U0126 (right column). All cells were treated with DMSO or U0126 for 24 h before re-seeding into low cluster plates. Cells were cultured in suspension for 24 h in the tissue culture incubator, after which images were taken with a camera attached to the Zeiss Axiovert microscope equipped with a x10 phase-contrast objective. C, Palladin mRNA and protein expression in untreated E1 cells, E1 cells treated with DMSO or U0126 for 24 h was studied using RT-PCR (a) and Western blot analysis (b), respectively. The expression of phosphorylated Erk (pErk) and total Erk in untreated E1 cells and E1 cells treated with DMSO or U0126 for 24 h was studied by Western blot analysis (b). Erk inhibition changed levels of pErk but not total Erk. GAPDH was used as a loading control. D, Confocal microscopy images of HCT116 cells, E1 cells treated with DMSO or 25 μM U126 for 24 h, fluorescently stained with palladin and E-cadherin antibodies.

Article Snippet: Polyclonal antibodies against palladin were purchased from Proteintech Group Inc., while those against total p44/42 MAP kinase (Erk1 and Erk2) and phospho-44/42 MAP kinase (Erk1 and Erk2) from Cell Signaling Technology.

Techniques: Inhibition, Expressing, Microscopy, Control, Suspension, Transfection, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Western Blot, Confocal Microscopy, Staining

Journal: International journal of oncology

Article Title: Palladin, an actin-associated protein, is required for adherens junction formation and intercellular adhesion in HCT116 colorectal cancer cells.

doi: 10.3892/ijo_00000742

Figure Lengend Snippet: Figure 6. Continued. E, RT-PCR analysis of palladin mRNA expression in HCT116 cells, E1 cells transfected with control RNAi or RNAi #2, followed by no treatment, or treatment with DMSO, 10 or 25 μM of U0126. Cells were treated with DMSO or U0126 for 24 h, at 24 h post-transfection. GAPDH was used as loading control. F, E1 cells transfected with control RNAi (GCctl), RNAi #2, #3 or #4, followed by treatment with DMSO, 10 or 25 μM of U0126. E1 cells were treated with DMSO or U0126 for 24 h. For RNAi- transfected E1 cells, DMSO or U0126 was added at 24 h post-transfection.

Article Snippet: Polyclonal antibodies against palladin were purchased from Proteintech Group Inc., while those against total p44/42 MAP kinase (Erk1 and Erk2) and phospho-44/42 MAP kinase (Erk1 and Erk2) from Cell Signaling Technology.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Control

Journal: International journal of oncology

Article Title: Palladin, an actin-associated protein, is required for adherens junction formation and intercellular adhesion in HCT116 colorectal cancer cells.

doi: 10.3892/ijo_00000742

Figure Lengend Snippet: Figure 7. Immunohistochemical analysis of palladin expression in matched normal colonic mucosa and colorectal tumors tissue sections. A, Immunohisto- chemical staining of palladin in the surface epithelium and crypt of normal colonic mucosa (left panel) (x100 magnification). A further magnified section of the surface epithelium (middle panel) (x400), and the crypt (right panel) (x400). B, Colorectal tumor section showing palladin staining at the central tumor area (a) and at the invasive front (d). The central tumor area has intact well-differentiated tubules, (b) (x400) shows the magnified section of the boxed region in (a) (x100). The palladin staining in central tumor area was compared to that of matched normal colonic mucosa (c) (x400). Region of the invasive front was shown in (d) (x40). The circled and boxed regions showed the moderately-/poorly-differentiated tumor tubules, which are respectively enlarged in (e) (x400) and (f) (x400). Cells that have dissociated from the tumor tubules at the invasive front are indicated by the arrow and boxed region in (d) and shown enlarged in C(a) (x200) and (b) (x200), respectively. C, Representative examples of palladin expression of de-differentiated tumor areas (indicated by arrows) in the invasive fronts of colorectal tumor sections, which could exhibit as dissociated cells that form small clusters (a), (b), (c) (x200), or isolated tumor cells (d) (x400). Brown staining indicates positive palladin staining, blue hematoxylin was used as the counterstain.

Article Snippet: Polyclonal antibodies against palladin were purchased from Proteintech Group Inc., while those against total p44/42 MAP kinase (Erk1 and Erk2) and phospho-44/42 MAP kinase (Erk1 and Erk2) from Cell Signaling Technology.

Techniques: Immunohistochemical staining, Expressing, Staining, Isolation