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1.
Figure 2

Figure 2. From: A Functional Interaction between the Survival Motor Neuron Complex and RNA Polymerase II.

Association of the SMN complex with RHA in vivo. Immunoprecipitation (IP) experiments were carried out on HeLa cell nucleoplasmic extract using anti-RHA or control rabbit preimmune antibodies. Immunoprecipitates were analyzed by SDS-PAGE and Western blotting with the antibodies to the indicated proteins. 10% of the input is shown.

Livio Pellizzoni, et al. J Cell Biol. 2001 January 8;152(1):75-86.
2.
Figure 6

Figure 6. From: A Functional Interaction between the Survival Motor Neuron Complex and RNA Polymerase II.

Specificity of the effects of SMNΔN27 expression. (A–I) Double-label confocal immunofluorescence experiments using anti-myc (A, D, and G) and either anti-hnRNP A1, anti-hnRNP C1/C2 or anti-SC35 antibodies (B, E, and H, respectively) on HeLa cells transiently transfected with mycSMNΔN27. The respective combined images are shown in C, F, and I. Dashed lines demarcate the nucleus. Bars, 5 μm.

Livio Pellizzoni, et al. J Cell Biol. 2001 January 8;152(1):75-86.
3.
Figure 3

Figure 3. From: A Functional Interaction between the Survival Motor Neuron Complex and RNA Polymerase II.

The SMN complex interacts with pol II CTD in vitro. (A) GST–CTD affinity chromatography. HeLa nuclear extract was incubated with either purified recombinant GST or GST–CTD, and bound proteins were analyzed by SDS-PAGE and Western blotting with antibodies to the indicated proteins. 2.5% of the input is shown. (B) RHA stimulates the interaction of SMN with pol II CTD. In vitro–translated [35S]methionine-labeled SMN, with (+) or without (−) in vitro–translated [35S]methionine-labeled RHA, was incubated with purified recombinant GST–CTD or GST as indicated. Binding reactions contained equal amounts of reticulocyte lysate. Bound proteins were analyzed by SDS-PAGE and autoradiography. 1% of the input is shown.

Livio Pellizzoni, et al. J Cell Biol. 2001 January 8;152(1):75-86.
4.
Figure 3

Figure 3. From: A Functional Interaction between the Survival Motor Neuron Complex and RNA Polymerase II.

The SMN complex interacts with pol II CTD in vitro. (A) GST–CTD affinity chromatography. HeLa nuclear extract was incubated with either purified recombinant GST or GST–CTD, and bound proteins were analyzed by SDS-PAGE and Western blotting with antibodies to the indicated proteins. 2.5% of the input is shown. (B) RHA stimulates the interaction of SMN with pol II CTD. In vitro–translated [35S]methionine-labeled SMN, with (+) or without (−) in vitro–translated [35S]methionine-labeled RHA, was incubated with purified recombinant GST–CTD or GST as indicated. Binding reactions contained equal amounts of reticulocyte lysate. Bound proteins were analyzed by SDS-PAGE and autoradiography. 1% of the input is shown.

Livio Pellizzoni, et al. J Cell Biol. 2001 January 8;152(1):75-86.
5.
Figure 5

Figure 5. From: A Functional Interaction between the Survival Motor Neuron Complex and RNA Polymerase II.

Expression of SMNΔN27 causes accumulation of RHA, TBP, and snRNPs in G/CBs. (A–F) Double-label confocal immunofluorescence experiments using anti-myc (A and D) and anti-RHA (B and E) antibodies on HeLa cells transiently transfected with mycSMNwt (A–C) and mycSMNΔN27 (D–F). (G–I) Double-label confocal immunofluorescence experiments using anti-myc (G) and anti-TBP (H) antibodies on HeLa cells transiently transfected with mycSMNΔN27. (J–L) Double-label confocal immunofluorescence experiments using anti-myc (J) and anti-Sm (K) antibodies on HeLa cells transiently transfected with mycSMNΔN27. The respective combined images are shown in C, F, I, and L. Colocalization results in a yellow signal. Dashed lines demarcate the nucleus. Bars, 5 μm.

Livio Pellizzoni, et al. J Cell Biol. 2001 January 8;152(1):75-86.
6.
Figure 4

Figure 4. From: A Functional Interaction between the Survival Motor Neuron Complex and RNA Polymerase II.

Expression of SMNΔN27 causes accumulation of pol IIa in G/CBs. (A–F) Double-label confocal immunofluorescence experiments using anti-myc (A and D), and anti-pol IIa (B and E) antibodies on HeLa cells transiently transfected with mycSMNwt (A–C) and mycSMNΔN27 (D–F). (G–L) Double-label confocal immunofluorescence experiments using anti-myc (G and J) and anti-pol IIo (H and K) antibodies on HeLa cells transiently transfected with mycSMNwt (G–I) and mycSMNΔN27 (J–L). The respective combined images are shown in C, F, I, and L. Colocalization results in a yellow signal. Dashed lines demarcate the nucleus. Bars, 5 μm.

Livio Pellizzoni, et al. J Cell Biol. 2001 January 8;152(1):75-86.
7.
Figure 8

Figure 8. From: A Functional Interaction between the Survival Motor Neuron Complex and RNA Polymerase II.

Expression of SMNΔN27 inhibits transcription by RNA pol I and II in vivo. (A–F) Double-label laser confocal microscopy of HeLa cells transiently transfected with mycSMNwt (A–C) and mycSMNΔN27 (D–F), showing the incorporation of BrU into RNA by indirect immunofluorescence using anti-myc (A and D) and anti-BrU (B and E) antibodies. BrU incorporation was performed as described in Materials and Methods on transfected cells before fixation and immunofluorescence staining. The respective combined images are shown in C and F. Colocalization results in a yellow signal. Dashed lines demarcate the nucleus. The nucleolus is indicated by an arrow in E. Bar, 5 μm.

Livio Pellizzoni, et al. J Cell Biol. 2001 January 8;152(1):75-86.
8.
Figure 7

Figure 7. From: A Functional Interaction between the Survival Motor Neuron Complex and RNA Polymerase II.

A rapid method for the in situ detection of transcription. (A) Indirect immunofluorescence detection of nascent RNA by laser confocal microscopy using anti-BrU antibodies on HeLa cells labeled in the presence 2 mM BrU as described in Materials and Methods. (B) Effect of RNase A treatment before immunostaining. (C) Effect of DNase I treatment before immunostaining. (D–E) Effect of low (0.04 μg/ml) or high (5 μg/ml) doses of actinomycin D (ActD) on BrU incorporation into RNA. (F) Effect of α-amanitin (50 μg/ml) on BrU incorporation into RNA. Dashed lines demarcate the nucleus. Bars, 5 μm.

Livio Pellizzoni, et al. J Cell Biol. 2001 January 8;152(1):75-86.
9.
Figure 1

Figure 1. From: A Functional Interaction between the Survival Motor Neuron Complex and RNA Polymerase II.

Identification of RHA as a novel SMN-interacting protein. (A) Coimmunoprecipitation experiments with anti-Gemin3 (11G9) or antitransportin (19D9) monoclonal antibodies were carried out from total HeLa cell extract. The immunoprecipitates (IP) were analyzed by SDS-PAGE and Coomassie blue staining. The band corresponding to the p130 protein coimmunoprecipitated with anti-Gemin3 antibodies was excised and identified by nanoelectrospray tandem mass spectrometry as described in Materials and Methods. Molecular weight markers, heavy (Ig h.c.) and light (Ig l.c.) chains of immunoglobulins, and the bands corresponding to the components of the SMN complex are indicated. (B) SMN interacts with RHA in vitro. In vitro–translated [35S]methionine-labeled RHA full length (RHA) was incubated with either GST, GST–Gemin2, or GST–SMN as indicated. 10% of the input is shown. (C) The SMN interaction with RHA is impaired by SMN mutations found in SMA patients and requires the RGG-rich domain of RHA. In vitro–translated [35S]methionine-labeled RHA full-length (RHA) or a COOH terminus deletion lacking the RGG domain (RHAΔRGG) were incubated with the indicated GST–Gemin2/SMN complexes containing either wild-type (wt) or mutant his-SMN proteins coexpressed and purified as described in Materials and Methods. 10% of the input is shown. (D) Schematic representation of the structure of RHA and the domains involved in the interaction with pol II, BRCA1, CBP, and SMN.

Livio Pellizzoni, et al. J Cell Biol. 2001 January 8;152(1):75-86.

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