rabbit anti-kai1  (Novus Biologicals)

Journal: Carcinogenesis

Article Title: Prognostic significance of KAI1/CD82 in human melanoma and its role in cell migration and invasion through the regulation of ING4.

doi: 10.1093/carcin/bgt346

Figure Lengend Snippet: Fig. 1. KAI1 expression is reduced in advanced human melanoma. (A–H) Representative images of KAI1 immunohistochemical staining in human melanocytic lesions. (A) and (E), strong KAI1 staining in CAN; (B) and (F), moderate KAI1 staining in DN; (C) and (G), weak KAI1 staining PM; D and H, negative KAI1 staining in MM. Bar = 50 μm. (I) KAI1 expression is significantly reduced when comparing common acquired nevi and dysplastic nevi (P = 0.04, χ2 test), dysplastic nevi and primary melanoma (P = 1.8 × 10-4, χ2 test) and primary melanoma and metastatic melanoma (P = 9.4 × 10−15, χ2 test). CAN, common acquired nevi; DN, dysplastic nevi; PM, primary melanoma; MM, metastatic melanoma.

Article Snippet: The primary rabbit anti-KAI1 antibody (1:1000 dilution, Novus Biologicals, Littleton, CO) and the biotin-labeled secondary antibody (DAKO Diagnostics, Glostrup, Denmark) were used.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Carcinogenesis

Article Title: Prognostic significance of KAI1/CD82 in human melanoma and its role in cell migration and invasion through the regulation of ING4.

doi: 10.1093/carcin/bgt346

Figure Lengend Snippet: Fig. 2. KAI1 expression is associated with 5 year survival of melanoma patients. Two hundred and sixty-two primary melanoma patients and 155 metastatic melanoma patients were analyzed. (A) Reduced KAI1 expression is correlated with poor overall and disease-specific 5 year survival in all melanoma patients (P = 1.2 × 10−7 and 3.6 × 10−7, respectively, log-rank test). (B) Reduced KAI1 expression is correlated with poor overall and disease-specific 5 year survival in primary melanoma patients (P = 0.003 and 0.007, respectively, log-rank test). (C) Reduced KAI1 expression is correlated with poor overall and disease-specific 5 year survival in metastatic melanoma patients (P = 0.012 and 0.015, respectively, log-rank test). Cum., cumulative.

Article Snippet: The primary rabbit anti-KAI1 antibody (1:1000 dilution, Novus Biologicals, Littleton, CO) and the biotin-labeled secondary antibody (DAKO Diagnostics, Glostrup, Denmark) were used.

Techniques: Expressing

Journal: Carcinogenesis

Article Title: Prognostic significance of KAI1/CD82 in human melanoma and its role in cell migration and invasion through the regulation of ING4.

doi: 10.1093/carcin/bgt346

Figure Lengend Snippet: Fig. 3. KAI1 regulates melanoma cell migration. (A, D) Western blot analysis of KAI1 expression. (A) Flag antibody was used and (D) KAI1 antibody was used. (B, E) Representative images of the effect of KAI1 overexpression or knockdown on melanoma cell migration. Twenty-four hours after transfection with si-KAI1-1, si-KAI1-2 or control siRNA, MMRU cells were transfected with KAI1 or control plasmids for 24 h. Then the monolayer of MMRU cells was scratched. The gaps were observed by microscopy and photographed. (C, F) The migrated cells during wound healing were counted randomly in five fields of each group, and each group was repeated three times. Columns, mean; bars, standard deviation. ***P < 0.001. Ctrl, control; CV, control vector; si, small interfering.

Article Snippet: The primary rabbit anti-KAI1 antibody (1:1000 dilution, Novus Biologicals, Littleton, CO) and the biotin-labeled secondary antibody (DAKO Diagnostics, Glostrup, Denmark) were used.

Techniques: Migration, Western Blot, Expressing, Over Expression, Knockdown, Transfection, Control, Microscopy, Standard Deviation, Plasmid Preparation

Journal: Carcinogenesis

Article Title: Prognostic significance of KAI1/CD82 in human melanoma and its role in cell migration and invasion through the regulation of ING4.

doi: 10.1093/carcin/bgt346

Figure Lengend Snippet: Fig. 4. KAI1 regulates stress fiber formation via ROCK. (A, C) The effect of KAI1 overexpression or knockdown on melanoma cell stress fiber formation. MMRU Cells were transfected with Flag-KAI1, control plasmid, si-KAI1-1, si-KAI1-2 or control siRNA, followed by serum starvation overnight and serum stimulation for 30 min. For ROCK inhibitor treatment, 10 μmol/l Y27632 in serum-free medium was added to the cells after serum starvation overnight and incubated for 2 h, and then the cells were incubated with complete medium containing 10% fetal bovine serum with 10 μmol/l Y27632 for 30 min. Magnification, ×400. (B, D) Quantitation of (A) and (C). RI, ROCK inhibitor Y27632. ***P < 0.001.

Article Snippet: The primary rabbit anti-KAI1 antibody (1:1000 dilution, Novus Biologicals, Littleton, CO) and the biotin-labeled secondary antibody (DAKO Diagnostics, Glostrup, Denmark) were used.

Techniques: Over Expression, Knockdown, Transfection, Control, Plasmid Preparation, Incubation, Quantitation Assay

Journal: Carcinogenesis

Article Title: Prognostic significance of KAI1/CD82 in human melanoma and its role in cell migration and invasion through the regulation of ING4.

doi: 10.1093/carcin/bgt346

Figure Lengend Snippet: Fig. 5. KAI1 regulates melanoma cell invasion and the activity of MMP-2. (A, C) The effect of KAI1 overexpression or knockdown on melanoma cell invasion using Transwell culture chamber. MMRU cells were transfected with Flag-KAI1, control plasmid, si-KAI1-1, si-KAI1-2 or control siRNA. The cells were seeded on to matrigel with serum-free medium, incubated for 24 h at 37°C, stained with crystal violet and quantified. (B, D) Quantitation of (A) and (C), respectively. The experiment was done in triplicate wells. (E) KAI1 inhibits the activity of MMP-2 in MMRU cells by performing the zymography assay. (F) Quantitation of (E). The experiment was repeated three times. ***P < 0.001.

Article Snippet: The primary rabbit anti-KAI1 antibody (1:1000 dilution, Novus Biologicals, Littleton, CO) and the biotin-labeled secondary antibody (DAKO Diagnostics, Glostrup, Denmark) were used.

Techniques: Activity Assay, Over Expression, Knockdown, Transfection, Control, Plasmid Preparation, Incubation, Staining, Quantitation Assay, Zymography

Journal: Carcinogenesis

Article Title: Prognostic significance of KAI1/CD82 in human melanoma and its role in cell migration and invasion through the regulation of ING4.

doi: 10.1093/carcin/bgt346

Figure Lengend Snippet: Fig. 6. Regulation of ING4 by KAI1 and its effect on melanoma cell migration. (A) Quantitative reverse transcription–PCR analysis was done by transfection cell with Flag-KAI1, control plasmid, si-KAI1-1, si-KAI1-2 or control siRNA. Expression of ING4 mRNAs was measured using real-time quantitative PCR and normalized with glyceraldehyde 3-phosphate dehydrogenase as loading control. (B) Western blot analysis of ING4 expression after the cells were transfected with Flag-KAI1, control plasmid, si-KAI1-1, si-KAI1-2 or control siRNA. (C) Western blot analysis of ING4 expression after cells were transfected with control siRNA and si-KAI1-1, si-p65 alone or together. (D) KAI1/p65/ING4 signaling pathway in melanoma migration. (E) The effect of reduced KAI1 expression on cell migration was reduced by forced ING4 expression. The cells were transfected with si-KAI1 or control siRNA, and then after 48 h, the cells were transfected with HA-ING4 or HA control plasmid. Monolayer MMRU cells were scratched and the gap was monitored by microscopy and photographed. (F) The reduction in cell migration by the forced KAI1 expression was compensated by ING4 knockdown. The cells were transfected with KAI1 or control plasmid, and then after 24 h, the cells were transfected with mi-ING4 or control plasmid. (G, H) Quantitation of (E) and (F), respectively. The migrated cells during wound healing were counted randomly in five fields of each group, and each group was repeated three times. Columns, mean; bars, standard deviation. ***P < 0.001. si, small interfering; Ctrl, control; HA, hemagglutinin; mi, microRNA.

Article Snippet: The primary rabbit anti-KAI1 antibody (1:1000 dilution, Novus Biologicals, Littleton, CO) and the biotin-labeled secondary antibody (DAKO Diagnostics, Glostrup, Denmark) were used.

Techniques: Migration, Reverse Transcription, Transfection, Control, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Microscopy, Knockdown, Quantitation Assay, Standard Deviation