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Results: 7

1.
Figure 7

Figure 7. A Road Map of APP Processing and Aβ Production. From: A Paired RNAi and RabGAP Overexpression Screen Identifies Rab11 as a Regulator of ?-Amyloid Production.

Integrated map of the Rabs identified in the screen as regulators of APP processing and Aβ production/secretion. Rabs indicated in green positively regulate Aβ levels, and those indicated in red are negative regulators. Note that the majority of the Rabs regulate trafficking to and from early endosomes.

Vinod Udayar, et al. Cell Rep. 2013 December 26;5(6):1536-1551.
2.
Figure 5

Figure 5. BACE1 Colocalizes with Rab11 Compartments, and Its Trafficking Is Altered upon Rab11 Dysfunction. From: A Paired RNAi and RabGAP Overexpression Screen Identifies Rab11 as a Regulator of ?-Amyloid Production.

(A) Live-cell images of COS cells transfected with BACE1-YFP or YFP-APP along with empty vector (control) or Rab11A DN. Expression of Rab11A DN protein is shown in the insets.
(B) COS cells were transiently cotransfected with BACE1-YFP (green) and empty vector, HA-tagged Rab11A WT or S25N DN mutant, and fixed and stained with HA (data not shown) and TfR antibodies (red) to label recycling endosomes. The colors in the first two panels are green for BACE1-YFP and red for TfR. Insets are the zoomed-in images of the corresponding images.
See also Figure S5A and Movies S1 and S2.

Vinod Udayar, et al. Cell Rep. 2013 December 26;5(6):1536-1551.
3.
Figure 1

Figure 1. A Multiplexing Platform for the Detection of Amyloidogenic Processing of APP. From: A Paired RNAi and RabGAP Overexpression Screen Identifies Rab11 as a Regulator of ?-Amyloid Production.

(A) Schematic of the screen.
(B) Cartoon of APP cleavage by β- and γ-secretases.
(C) Cartoon of ECL detection system.
(D) Incubation of synthetic Aβ (Aβ40, black) and recombinant sAPPβ (red) either individually or together gives specific signals.
(E) Supernatants from HEK (HEK-sweAPP) or HeLa cells overexpressing the Swedish mutant of APP (HeLa-sweAPP) and primary cortical neurons from Arc/sweAPP Tg analyzed for Aβ (black) and sAPPβ (white for HEK and HeLa; gray for the cortical neurons) levels.
(F and G) Specificity of the ECL-multiplex assay platform. Signals for Aβ40 (black) and WT sAPPβ (gray) from conditioned medium of HEK-WTAPP (F) or HEK-sweAPP (G) analyzed with capture antibodies specific for Aβ and WT sAPPβ.
(H and I) Signals for Aβ40 (black) and swe-sAPPβ (gray) from conditioned medium of HEK-WTAPP cells (H) or HEK-sweAPP cells (I).
Error bars indicate SD. See also Figures S1A and S1B.

Vinod Udayar, et al. Cell Rep. 2013 December 26;5(6):1536-1551.
4.
Figure 3

Figure 3. RabGAP Screen Identifies GAPs and Rabs in the Regulation of Aβ and sAPPβ Levels. From: A Paired RNAi and RabGAP Overexpression Screen Identifies Rab11 as a Regulator of ?-Amyloid Production.

(A) Graph showing the levels of Aβ (black) and sAPPβ (gray) after RabGAP plasmid overexpression. HeLa-sweAPP cells were transfected with plasmid expressing RabGAP fused with GFP or a plasmid expressing GFP (negative control) and assayed for Aβ and sAPPβ. Error bars indicate SEM.
(B) Graphs showing Z scores of the effects on sAPPβ and Aβ levels after RabGAP overexpression.
(C) Table showing RabGAPs that had the strongest effect on Aβ and/or sAPPβ levels along with their cognate Rab(s). The asterisk (*) indicates that the TBC1D14 is not a GAP for Rab11 but has been shown to perturb the localization and function of Rab11 (Longatti et al., 2012).
See also Figure S1D.

Vinod Udayar, et al. Cell Rep. 2013 December 26;5(6):1536-1551.
5.
Figure 2

Figure 2. RNAi Screen of Genome-wide Rabs Identifies Rabs Involved in Regulating the Levels of Aβ and sAPPβ. From: A Paired RNAi and RabGAP Overexpression Screen Identifies Rab11 as a Regulator of ?-Amyloid Production.

(A and B) Graph showing the levels of Aβ (A) and sAPPβ (B) from the Rab siRNA screen. HeLa-sweAPP cells were transfected with Scrambled oligo (negative control), or pooled siRNA against APP, BACE1, PEN2, and the 60 Rabs and assayed for Aβ and sAPPβ. Error bars indicate SEM.
(C) List of genes that had the strongest effect on Aβ and sAPPβ levels after siRNA-mediated silencing along with the Z score and t test values.
(D) 2D plot representing the levels of Aβ and sAPPβ from the Rabs siRNA screen. Positive controls APP, BACE1, and PEN2 are indicated in green, and negative control Scrambled is indicated in blue. Rabs silencing that led to the strongest decrease in both Aβ and sAPPβ is indicated in red. All statistics were performed using the two-tailed t tests and one-way ANOVA (Table S4).
See also Figures S1C, S2, and S3.

Vinod Udayar, et al. Cell Rep. 2013 December 26;5(6):1536-1551.
6.
Figure 6

Figure 6. Rab11 Regulates the Surface Recycling of BACE1 and Is Essential for Maintenance of BACE1 Cell Surface Levels. From: A Paired RNAi and RabGAP Overexpression Screen Identifies Rab11 as a Regulator of ?-Amyloid Production.

(A) HeLa-swAPP cells were transfected with either control plasmids or the DN mutant of Rab11A and Flag-tagged BACE1. Cell surface levels of BACE1 were determined via FACS on nonpermeabilized cells using FLAG antibodies and Alexa 647-coupled secondary antibodies.
(B) The mean and the median intensity of the cell surface levels of BACE1 are lower in Rab11A DN-expressing cells.
(C) Microscopy of cell surface BACE1 on nonpermeabilized HeLa cells expressing BACE1-Flag. Rab11A DN-expressing cells have marked decrease in cell surface BACE1.
(D) Immunofluorescence of cell surface BACE1 on nonpermeabilized primary neurons transfected with BACE1-Flag. Also in neurons, Rab11A DN-expressing cells have a marked decrease in cell surface BACE1.
(E) Recycling of BACE1 is significantly decreased in Rab11A DN-expressing cells. Cells were transfected with BACE-FLAG/Rab11A constructs (WT and DN) and incubated with Alexa 647-conjugated M1 FLAG primary antibodies for 30 min at 37°C, followed by washing and incubation with Alexa 488 secondary antibodies at 37°C for 30 min. This allows the primary antibody to bind the surface/cycling BACE1 and label the endosomal BACE population, and the secondary antibody to only bind the BACE1 that recycled to the surface. A ratio of the secondary to the primary gives the amount recycled in this time.
See also Figures S4F and S5B.

Vinod Udayar, et al. Cell Rep. 2013 December 26;5(6):1536-1551.
7.
Figure 4

Figure 4. Rab11 Regulates BACE1 Cleavage of APP in Model Cell Lines and Primary Neurons from WT and APP Transgenic Mice. From: A Paired RNAi and RabGAP Overexpression Screen Identifies Rab11 as a Regulator of ?-Amyloid Production.

(A) HeLa-sweAPP cells were transfected with scrambled oligo (MEDGC, negative control) or pooled siRNA against APP and Rab11A and assayed for Aβ (dark gray) and sAPPβ (light gray). Error bars indicate SD.
(B) HeLa-sweAPP cells were transfected with scrambled oligo or pooled siRNA against Rab11A, and the lysates were assayed by western blotting as indicated.
(C) HeLa-sweAPP cells were transfected with scrambled oligo or pooled siRNAs against APP and Rab11A and assayed for Aβ38 (light gray), Aβ40 (dark gray), and Aβ42 (black). Error bars indicate SEM.
(D) HeLa-sweAPP cells were transfected with scrambled oligo or pooled siRNA against APP, single siRNA (si1–si4) against Rab11A, and pooled siRNA against Rab11A and assayed for Aβ40 (dark gray) and cell viability (light gray), and the lysates were assayed by western blotting as indicated (inset). Error bars indicate SD.
(E) HeLa-sweAPP cells were transfected with PCDNA 3.1 (negative control), Rab11A DN, and Rab11B DN and assayed for Aβ40 (dark gray) and sAPPβ (light gray). Error bars indicate SEM.
(F) HeLa-sweAPP cells were transfected with scrambled oligo or pooled siRNA against APP, BACE1, and Rab11FIP1–Rab11FIP5 and assayed for sAPPβ. Error bars indicate SEM.
(G–J) Silencing of Rab11 in primary neurons from APP transgenic and WT mice reduces β cleavage of APP and Aβ levels.
(G) Rab11A and Rab11B were silenced in primary cortical and hippocampal neurons isolated from APP transgenic mice expressing the Swedish mutation in APP, and the secreted levels of Aβ and sAPPβ were measured. The scrambled oligo was used as a negative control and siRNA targeting APP as positive control. Error bars indicate SD.
(H) Western blotting with Rab11A and GAPDH antibodies from control and Rab11-silenced primary neurons.
(I) Rab11A and Rab11B were silenced in primary cortical and hippocampal neurons isolated from WT mice, and the secreted levels of endogenous Aβ40 and Aβ42 were measured. The scrambled oligo was used as a negative control and siRNA targeting APP as positive control. Error bars indicate SD.
(J) Western blotting with Rab11A and GAPDH antibodies from control and Rab11-silenced primary WT neurons. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with scrambled MEDGC or PCDNA3.1. n.s., not significant. All statistics were performed using the two-tailed t tests. Similar significance was also observed with one-way ANOVA (Dunnett’s a posteriori analysis) (Table S5). See also Figures S4A–S4E.

Vinod Udayar, et al. Cell Rep. 2013 December 26;5(6):1536-1551.

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