Journal: Investigative Ophthalmology & Visual Science

Article Title: Vitreous Humor Changes Expression of Iron-Handling Proteins in Lens Epithelial Cells

doi: 10.1167/iovs.16-20610

Figure Lengend Snippet: ( A ) Phase contrast image of control (C) and vitreous-treated LEC (V). LEC were treated for 96 hours with DMEM containing 1% FBS and 33% of vitreous humor. Control LEC were grown in DMEM containing 1% FBS without vitreous humor. ( B ) Western blot analysis of aquaporin 0 expression in treated (V) and control LEC (C). Lens lysates containing 50 μg proteins were separated by 12% SDS-PAGE, transferred to nitrocellulose membranes, and probed with rabbit anti-aquaporin 0 polyclonal antibodies. Canine lens was dissected and the section containing lens fibers was sonicated. Fifty micrograms of protein sample of sonicated lens fiber cells was used as a positive control (St). Blot was evaluated with ChemiDoc MP Imaging System.

Article Snippet: Proteins were semidry transferred to nitrocellulose membranes (Hybond ECL; GE Healthcare, Pittsburg, PA, USA) using the transfer Trans-Blot SD Cell (Bio-Rad Laboratories, Richmond, CA, USA) system.

Techniques: Control, Western Blot, Expressing, SDS Page, Sonication, Positive Control, Imaging

Journal: Investigative Ophthalmology & Visual Science

Article Title: Vitreous Humor Changes Expression of Iron-Handling Proteins in Lens Epithelial Cells

doi: 10.1167/iovs.16-20610

Figure Lengend Snippet: Western blot analysis of ferritin L-chain in the lysates of control LEC (C), vitreous-treated LEC (V), and sonicated lens fiber cells (Fib). Protein samples, 35 to 50 μg, were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system. Proteins were transferred to nitrocellulose membranes and probed with antibodies specific to ferritin L-chain. Purified canine heart ferritin was used as standard (St).

Article Snippet: Proteins were semidry transferred to nitrocellulose membranes (Hybond ECL; GE Healthcare, Pittsburg, PA, USA) using the transfer Trans-Blot SD Cell (Bio-Rad Laboratories, Richmond, CA, USA) system.

Techniques: Western Blot, Control, Sonication, SDS Page, Purification

Journal: Investigative Ophthalmology & Visual Science

Article Title: Vitreous Humor Changes Expression of Iron-Handling Proteins in Lens Epithelial Cells

doi: 10.1167/iovs.16-20610

Figure Lengend Snippet: Western blot analysis of ferritin H- and L-chains in the lysates of control (C) and vitreous-treated LEC (V). Confluent LEC were cultured in DMEM with 10% FBS with (V) or without (C) 33% (vol/vol) of vitreous humor for 24 or 96 hours. LEC lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified canine heart ferritin was used as standard (St) for ferritin chains. Blot was reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The significance of differences was determined by paired t -test. Data represent the mean ± SEM; n = 11; statistically different from corresponding C, * P < 0.05.

Article Snippet: Proteins were semidry transferred to nitrocellulose membranes (Hybond ECL; GE Healthcare, Pittsburg, PA, USA) using the transfer Trans-Blot SD Cell (Bio-Rad Laboratories, Richmond, CA, USA) system.

Techniques: Western Blot, Control, Cell Culture, SDS Page, Purification, Imaging

Journal: Investigative Ophthalmology & Visual Science

Article Title: Vitreous Humor Changes Expression of Iron-Handling Proteins in Lens Epithelial Cells

doi: 10.1167/iovs.16-20610

Figure Lengend Snippet: Western blot analysis of TfR1 in control (C)- and vitreous-treated LEC (V). Confluent LEC were cultured in DMEM with 10% FBS with or without 33% (vol/vol) of vitreous humor for 24 or 96 hours. LEC lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified human placenta TfR1 was used as a standard (St) for canine TfR1. TfR1 was immunodetected with monoclonal mouse anti-human TfR1 antibodies. Blot was reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The significance of differences was determined by paired t -test. Data represent the mean ± SEM; n = 9; statistically different from corresponding C, * P < 0.05; statistically different from corresponding V 24 hours, ∧ P < 0.05.

Article Snippet: Proteins were semidry transferred to nitrocellulose membranes (Hybond ECL; GE Healthcare, Pittsburg, PA, USA) using the transfer Trans-Blot SD Cell (Bio-Rad Laboratories, Richmond, CA, USA) system.

Techniques: Western Blot, Control, Cell Culture, SDS Page, Purification, Imaging

Journal: Investigative Ophthalmology & Visual Science

Article Title: Vitreous Humor Changes Expression of Iron-Handling Proteins in Lens Epithelial Cells

doi: 10.1167/iovs.16-20610

Figure Lengend Snippet: Effects of DFO on expression of ferritin chains ( A ) and TfR1 ( B ) in LEC treated with vitreous humor (V) and control LEC (C). LEC were treated with 100 μM DFO (C/DFO;V/DFO) for 24 hours in serum-free DMEM without (C) or with 33% vitreous humor (V). Cell lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified canine heart ferritin was used as standard (St) for ferritin chains. Purified human placenta TfR1 was used as a standard (St) for canine TfR1. After Western transfer to a nitrocellulose membrane, ferritin chains were immunodetected with canine chain–specific custom-made antibodies. TfR1 was immunodetected with monoclonal mouse anti-human TfR1 antibodies. Blots were reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The significance of differences was determined by paired t -test. Data represent the mean ± SEM; n = 4; statistically different from C, * P < 0.05; statistically different from C/DFO, ∧ P < 0.05.

Article Snippet: Proteins were semidry transferred to nitrocellulose membranes (Hybond ECL; GE Healthcare, Pittsburg, PA, USA) using the transfer Trans-Blot SD Cell (Bio-Rad Laboratories, Richmond, CA, USA) system.

Techniques: Expressing, Control, SDS Page, Purification, Western Blot, Membrane, Imaging

Journal: Investigative Ophthalmology & Visual Science

Article Title: Vitreous Humor Changes Expression of Iron-Handling Proteins in Lens Epithelial Cells

doi: 10.1167/iovs.16-20610

Figure Lengend Snippet: Effects of cycloheximide on expression of ferritin chains ( A ) and TfR1 ( B ) in LEC treated with vitreous humor (V) and control LEC (C). LEC were treated with 10 μM cycloheximide for 24 hours in DMEM containing 10% FBS without (Chx) or with 33% vitreous humor (V/Chx). Cell lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified canine heart ferritin was used as standard (St) for ferritin chains. Purified human placenta TfR1 was used as a standard (St) for canine TfR1. After Western transfer to a nitrocellulose membrane, ferritin chains were immunodetected with canine chain–specific custom-made antibodies. TfR1 was immunodetected with monoclonal mouse anti-human TfR1 antibodies. Blots were reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The significance of differences was determined by using Tukey's HSD test. Data represent the mean ± SEM; n = 3; statistically different from corresponding C, * P < 0.05; statistically different from corresponding V, ∧ P < 0.05.

Article Snippet: Proteins were semidry transferred to nitrocellulose membranes (Hybond ECL; GE Healthcare, Pittsburg, PA, USA) using the transfer Trans-Blot SD Cell (Bio-Rad Laboratories, Richmond, CA, USA) system.

Techniques: Expressing, Control, SDS Page, Purification, Western Blot, Membrane, Imaging

Journal: Investigative Ophthalmology & Visual Science

Article Title: Vitreous Humor Changes Expression of Iron-Handling Proteins in Lens Epithelial Cells

doi: 10.1167/iovs.16-20610

Figure Lengend Snippet: Effects of actinomycin D on expression of ferritin chains ( A ) and TfR1 ( B ) in LEC treated with vitreous humor (V) and control LEC (C). LEC were treated with 1 μg/1 mL actinomycin D for 24 hours in DMEM containing 10% FBS without (ActD) or with 33% vitreous humor (V/ActD). Cell lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified canine heart ferritin was used as standard (St) for ferritin chains. Purified human placenta TfR1 was used as a standard (St) for canine TfR1. After Western transfer to a nitrocellulose membrane, ferritin chains were immunodetected with canine chain–specific custom-made antibodies. TfR1 was immunodetected with monoclonal mouse anti-human TfR1 antibodies. Blots were reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The significance of differences was determined by using Tukey's HSD test. Data represent the mean ± SEM; n = 3; statistically different from corresponding C, * P < 0.05; statistically different from corresponding V, ∧ P < 0.05.

Article Snippet: Proteins were semidry transferred to nitrocellulose membranes (Hybond ECL; GE Healthcare, Pittsburg, PA, USA) using the transfer Trans-Blot SD Cell (Bio-Rad Laboratories, Richmond, CA, USA) system.

Techniques: Expressing, Control, SDS Page, Purification, Western Blot, Membrane, Imaging

Journal: Investigative Ophthalmology & Visual Science

Article Title: Vitreous Humor Changes Expression of Iron-Handling Proteins in Lens Epithelial Cells

doi: 10.1167/iovs.16-20610

Figure Lengend Snippet: Western blot analysis of ferritin H- and L-chains in the lysates of LEC treated with differentially processed fractions of vitreous humor. LEC were treated for 24 hours without (C) or with 33% of vitreous humor (V) and with equivalent amount of differentially processed vitreous fractions, in DMEM containing 10% FBS. Vf, vitreous humor filtered through glass wool; Va, acetone precipitate of Vf dissolved in complete DMEM; Vmf, Microcon 10 filtrate of Vf ; Vmr, Microcon 10 retentate of Vf; Vb, Vf boiled for 10 minutes. Cell lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified canine heart ferritin was used as standard (St) for ferritin chains. After Western transfer to a nitrocellulose membrane, ferritin chains were immunodetected with canine chain–specific custom-made antibodies. Blots were reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The blot shown is representative of two experiments.

Article Snippet: Proteins were semidry transferred to nitrocellulose membranes (Hybond ECL; GE Healthcare, Pittsburg, PA, USA) using the transfer Trans-Blot SD Cell (Bio-Rad Laboratories, Richmond, CA, USA) system.

Techniques: Western Blot, SDS Page, Purification, Membrane, Control, Imaging

Journal: Investigative Ophthalmology & Visual Science

Article Title: Vitreous Humor Changes Expression of Iron-Handling Proteins in Lens Epithelial Cells

doi: 10.1167/iovs.16-20610

Figure Lengend Snippet: Effects of treatment of LEC with vitreous humor collected from dogs 7 to 10 years old with (Vcat) or without (V) age-related cataract on expression of ferritin chains and TfR1. LEC were treated with 33% vitreous humor in DMEM containing 10% FBS for 96 hours. Cells nontreated with vitreous humors were used as controls (C). Cell lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified canine heart ferritin was used as standard (St) for ferritin chains, and purified human placenta TfR1 was used as a standard (St) for canine TfR1. After Western transfer to a nitrocellulose membrane, ferritin chains were immunodetected with canine chain–specific custom-made antibodies. TfR1 was immunodetected with monoclonal mouse anti-human TfR1 antibodies. Blots were reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The significance of differences was determined by paired t -test. Data represent the mean ± SEM; n = 7; statistically different from corresponding V, * P < 0.05.

Article Snippet: Proteins were semidry transferred to nitrocellulose membranes (Hybond ECL; GE Healthcare, Pittsburg, PA, USA) using the transfer Trans-Blot SD Cell (Bio-Rad Laboratories, Richmond, CA, USA) system.

Techniques: Expressing, SDS Page, Purification, Western Blot, Membrane, Control, Imaging