Journal: Oncotarget

Article Title: The role of prospero homeobox 1 (PROX1) expression in follicular thyroid carcinoma cells

doi: 10.18632/oncotarget.23167

Figure Lengend Snippet: FTC-133 cells were treated with LY204002 (A) , the PI3K inhibitor (50μM), or with A6730 (B) , the AKT1/AKT2 inhibitor (10 μM), for 48 hours. Negative control cells were grown with DMSO instead of the inhibitors. (C) AKT and phospho-AKT expression in FTC-133 cells after silencing of PROX1 . The whole cell protein extracts were prepared and analyzed by Western blotting using a primary antibody against phospho-AKT (Ser473), AKT, or Prox1, followed by an incubation with relevant goat anti-rabbit or goat-anti mouse HRP-conjugated affinity-purified secondary antibodies. β-actin was used as a loading control.

Article Snippet: Preparation of total protein extracts from whole cells and Western blotting were carried out as previously described using the following antibodies: goat antibody against human Pro2-Gln259 Prox1 (AF2727, R&D Systems, Minneapolis, MN USA) diluted 1:2000 or rabbit anti-human against Prox1 (Cell Signaling #13910) diluted 1:1000 as primary antibodies, followed by a HRP-conjugated secondary antibody: a rabbit anti-goat secondary antibody (Jackson ImmunoResearch Laboratories, Vest Grow, Pa, USA) diluted 1:20,000 or a goat anti-rabbit antibody (DAKO, Carpinteria, CA, USA) diluted 1:5000, depending on the origin of the primary antibody [ ].

Techniques: Negative Control, Expressing, Western Blot, Incubation, Affinity Purification, Control

Journal: Oncotarget

Article Title: The role of prospero homeobox 1 (PROX1) expression in follicular thyroid carcinoma cells

doi: 10.18632/oncotarget.23167

Figure Lengend Snippet: PROX1 expression in FTC-133 cells following PROX1 silencing with PROX1 -specific siRNA (A–C) or PROX1 overexpression (D–F). siNEG: negative control (cells transfected with negative siRNA). (A) and (D) PROX1 mRNA levels determined by real-time quantitative PCR (RT-qPCR). The results were normalized against ACTB transcript levels and bars represent the average PROX1 transcript levels in cells transfected with PROX1 -siRNA compared to control cells. The results represent means from five independent experiments. (B) and (E) Prox1 protein levels determined by Western blotting, and (C and F) by immunofluorescent staining. Prox1 was detected with a rabbit anti-Prox1 polyclonal antibody and the DyLight 594-conjugated purified rabbit F(ab) 2 anti-goat secondary antibodies (red), and the nuclei were stained with DAPI (blue).

Article Snippet: Preparation of total protein extracts from whole cells and Western blotting were carried out as previously described using the following antibodies: goat antibody against human Pro2-Gln259 Prox1 (AF2727, R&D Systems, Minneapolis, MN USA) diluted 1:2000 or rabbit anti-human against Prox1 (Cell Signaling #13910) diluted 1:1000 as primary antibodies, followed by a HRP-conjugated secondary antibody: a rabbit anti-goat secondary antibody (Jackson ImmunoResearch Laboratories, Vest Grow, Pa, USA) diluted 1:20,000 or a goat anti-rabbit antibody (DAKO, Carpinteria, CA, USA) diluted 1:5000, depending on the origin of the primary antibody [ ].

Techniques: Expressing, Over Expression, Negative Control, Transfection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Control, Western Blot, Staining, Purification

Journal: Oncotarget

Article Title: The role of prospero homeobox 1 (PROX1) expression in follicular thyroid carcinoma cells

doi: 10.18632/oncotarget.23167

Figure Lengend Snippet: Fixed and permeabilized FTC-133 cells depleted of Prox1 or transfected with siNEG RNA were incubated overnight at 4°C with appropriate primary antibodies, followed by a one-hour room-temperature incubation with the appropriate secondary Rhodamine (TRICT)-conjugated AffiniPure F(ab’) 2 fragment goat anti-rabbit IgG and DyLight549-conjugated AffiniPure goat anti-mouse IgG antibodies (red). Images are representative confocal micrographs of FTC-133/si PROX1 cells in which F-actin was stained with phalloidin-AlexaFluor488 (green) and the nuclei (DNA) with DAPI (blue), taken at a 630x magnification. The yellow boxes depict the zoomed regions in the corresponding images.

Article Snippet: Preparation of total protein extracts from whole cells and Western blotting were carried out as previously described using the following antibodies: goat antibody against human Pro2-Gln259 Prox1 (AF2727, R&D Systems, Minneapolis, MN USA) diluted 1:2000 or rabbit anti-human against Prox1 (Cell Signaling #13910) diluted 1:1000 as primary antibodies, followed by a HRP-conjugated secondary antibody: a rabbit anti-goat secondary antibody (Jackson ImmunoResearch Laboratories, Vest Grow, Pa, USA) diluted 1:20,000 or a goat anti-rabbit antibody (DAKO, Carpinteria, CA, USA) diluted 1:5000, depending on the origin of the primary antibody [ ].

Techniques: Transfection, Incubation, Staining

Journal: Oncotarget

Article Title: The role of prospero homeobox 1 (PROX1) expression in follicular thyroid carcinoma cells

doi: 10.18632/oncotarget.23167

Figure Lengend Snippet: Primary and secondary antibodies used to analyse the expression of Prox1 and other proteins by western blotting and immunochistochemistry

Article Snippet: Preparation of total protein extracts from whole cells and Western blotting were carried out as previously described using the following antibodies: goat antibody against human Pro2-Gln259 Prox1 (AF2727, R&D Systems, Minneapolis, MN USA) diluted 1:2000 or rabbit anti-human against Prox1 (Cell Signaling #13910) diluted 1:1000 as primary antibodies, followed by a HRP-conjugated secondary antibody: a rabbit anti-goat secondary antibody (Jackson ImmunoResearch Laboratories, Vest Grow, Pa, USA) diluted 1:20,000 or a goat anti-rabbit antibody (DAKO, Carpinteria, CA, USA) diluted 1:5000, depending on the origin of the primary antibody [ ].

Techniques: Expressing, Western Blot, Affinity Purification, Purification, Control, Immunofluorescence