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Results: 6

1.
Figure 4

Figure 4. From: The Human Ubiquitin Conjugating Enzyme, UBE2E3, Is Required for Proliferation of Retinal Pigment Epithelial Cells.

UBE2E3 knockdown causes a dramatic elevation of p27Kip1 levels. (A) Representative FACS data of cells treated with siCON siRNA (left) or UBE2E3-specific siRNA duplexes 1 to 4 (Si2E3, right). Two days post-siRNA treatment, cells were colabeled with an anti-p27Kip1 antibody and with propidium iodide. The diagonal line across each graph is identically positioned and permits a comparison of the location of the dots between the two graphs. (B) Representative Western blots from the samples used for FACS in (A). Top: increase in p27Kip1 levels in response to UBE2E3 knockdown; middle: si2E3 decreased UBE2E3 levels below the detection limit of α-2E3. The β-tubulin blot illustrates that equal amounts of lysate were loaded in each lane.

Kendra S. Plafker, et al. Invest Ophthalmol Vis Sci. ;49(12):5611-5618.
2.
Figure 1

Figure 1. From: The Human Ubiquitin Conjugating Enzyme, UBE2E3, Is Required for Proliferation of Retinal Pigment Epithelial Cells.

α-2E3 specifically recognizes UBE2E3, and the enzyme localizes to interphase nuclei in telomerase-immortalized retinal pigment epithelial cells (RPE-1s). (A) α-GST (left) and α-2E3 (right) Western blot of GST and GST-E2 fusion proteins. Ten nanograms of each protein was loaded per lane. (B) Western blot with α-2E3 of solubilized RPE-1 cell lysate (left) and a lysate derived from mouse retina (right). Thirty micrograms of RPE-1 cell lysate and 25 μg of mouse retina lysate were analyzed. The migration of molecular weight markers is shown to the left of blots (A) and (B). UbcM2 is the mouse counterpart of UBE2E3. (C) Representative photomicrographs of RPE-1 cells immunostained with α-2E3 (right). DNA was counterstained with DAPI (left); white arrows: cells in mitosis. Images were captured with a 20× phase objective. Scale bar, 10 μm.

Kendra S. Plafker, et al. Invest Ophthalmol Vis Sci. ;49(12):5611-5618.
3.
Figure 6

Figure 6. From: The Human Ubiquitin Conjugating Enzyme, UBE2E3, Is Required for Proliferation of Retinal Pigment Epithelial Cells.

UbcM2, the mouse counterpart of UBE2E3, is downregulated during RPE maturation in the developing mouse eye. (A) Representative photomicrographs of X-Gal-labeled cryosections from E13 embryos (Aa, Ab) and P17 mice (Ac, Ad). (Ab, Ad) Wild-type samples; (Aa, Ac) samples from UbcM2 heterozygous mice. WT, wild type; UNP, undifferentiated retinal precursors; OS, outer segments; IS, inner segments; ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. (B) Graph of X-Gal spots counted per mm of RPE from an average of 10 sections from each of two animals for each time point. Error bars, SEM. *Statistically significant, according to a two-tailed distribution, two-sample, equal-variance Student's t-test with P < 0.0002.

Kendra S. Plafker, et al. Invest Ophthalmol Vis Sci. ;49(12):5611-5618.
4.
Figure 2

Figure 2. From: The Human Ubiquitin Conjugating Enzyme, UBE2E3, Is Required for Proliferation of Retinal Pigment Epithelial Cells.

Knockdown of UBE2E3 by siRNA causes a block of cell proliferation. (A) RNA was isolated from cells treated with duplexes (Du) 1 to 4 or with a mixture of the duplexes (Du 1+2+3+4). RT-PCR products were generated with UBE2E3- and importin-11-specific primers. (B) α-2E3 Western blot demonstrating UBE2E3 knockdown by each of the UBE2E3-specific siRNAs. The cells were harvested 2 days after siRNA treatment and lysates derived from 100,000 cells were loaded in each lane. Left: migration of molecular weight markers. (C) Graph of data obtained from cell counts on the indicated days after siRNA treatment. The graph represents averages obtained from three independent experiments. Error bars SD. NB, The error bar for the day 3 si2E3 bar is not detectable in this graph because of the close grouping of the data points from the three independent experiments (180,900, 189,000, and 182,500 cells).

Kendra S. Plafker, et al. Invest Ophthalmol Vis Sci. ;49(12):5611-5618.
5.
Figure 3

Figure 3. From: The Human Ubiquitin Conjugating Enzyme, UBE2E3, Is Required for Proliferation of Retinal Pigment Epithelial Cells.

UBE2E3 knockdown causes RPE-1 cells to exit from the cell cycle and flatten. (A) Immunofluorescence analysis of RPE-1 cells treated with either siCON or UBE2E3-specific siRNA (duplexes 2 and 3). Two days after siRNA treatment, the cells were colabeled with antibodies against p27Kip1 (center column) and Ki-67 (right column). DNA was counterstained with DAPI (left column). (B) Depletion of UBE2E3 caused RPE-1 cells to spread and flatten. The cells were imaged live with a 20× phase objective. Below the photomicrographs is a graph of average cell area (measured in pixels) for the indicated treatments. Data represent the pooled areas of 93 cells (siCon), 95 cells (duplex 2), and 92 cells (duplex 3) from two independent experiments. *Statistically significant, according to a two-tailed distribution, two-sample, equal-variance Student's t-test with P < 0.00001. Scale bar, 32.6 μm.

Kendra S. Plafker, et al. Invest Ophthalmol Vis Sci. ;49(12):5611-5618.
6.
Figure 5

Figure 5. From: The Human Ubiquitin Conjugating Enzyme, UBE2E3, Is Required for Proliferation of Retinal Pigment Epithelial Cells.

The elevation of p27Kip1 levels by UBE2E3 depletion can be rescued by reintroduction of the enzyme. (A) α-2E3 Western blots of lysates derived from RPE-1 cells transfected with plasmids expressing RFP-H2B (Mock) (lane 1) or untagged UBE2E3 (lane 2). The samples in the right blot were also subjected to treatment with UBE2E3-specific siRNA duplexes 1 to 4 (lanes 3 and 4). Asterisk: an α-2E3 reactive band that is produced by overexpression of the enzyme (lanes 2 and 4). The high molecular weight species also result from overexpression of the enzyme (lanes 2 and 4) and likely represent auto-ubiquitylated enzyme. (B) RPE-1 cells were transfected with plasmids encoding either RFP-H2B mRNA (mock; Ba, Bb, Be, Bf) or siRNA-impervious UBE2E3 mRNA (Bc, Bd, Bg, Bh). The following day, the cells were treated with either siCON (Bb, Bd, Bf, Bh) or si2E3 duplexes 1 to 4 (Ba, Bc, Be, Bg). Three days later, the cells were processed for anti-p27Kip1 immunofluorescence and DAPI staining. Representative photomicrographs for each of the conditions tested are shown. (Bad) DAPI-stained cells; (Beh) the corresponding anti-p27Kip1 immunofluorescence. Scale bar, 10 μm. (C) The percent relative pixel intensity of nuclear p27Kip1 per cell was quantitated from a series of photomicrographs and plotted in ascending order for 250 cells/sample. Cells were treated with si2E3 and mock rescue, si2E3 and UBE2E3 rescue, siCON and mock rescue, or siCON and UBE2E3 rescue.

Kendra S. Plafker, et al. Invest Ophthalmol Vis Sci. ;49(12):5611-5618.

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