Journal:

Article Title: In Cultured Oligodendrocytes the A/B-type hnRNP CBF-A Accompanies MBP mRNA Bound to mRNA Trafficking Sequences

doi: 10.1091/mbc.E07-10-1083

Figure Lengend Snippet: CBF-A, hnRNP A2, hnRNP A3, and hnRNP U are part of the same multiprotein complex. (A) Specificity of the affinity-purified peptide specific polyclonal anti-CBF-A antibody. Total protein extracts from HeLa cells were resolved by SDS-PAGE, blotted, and stained with Coomassie Blue (lane 1), immunostained with the CBF-A preimmune serum (lane 2), or with the affinity-purified anti-CBF-A antibody (lane 3) and with antibody SAK22 recognizing both CBF-A isoforms p37 and p42 (lane 4). (B) Sucrose gradient analysis of CBF-A, hnRNP A2, and hnRNP A3 from HeLa nuclear extracts. Fractions were resolved by SDS/PAGE and analyzed on immunoblots with antibodies to CBF-A and hnRNP A2/A3. (C) Schematic representation of recombinant hnRNP A2 and A3 and CBF-A constructs. (D) Pulldown experiment using S-tagged hnRNP A2, S-tagged hnRNP A3, or GST-tagged CBF-A constructs. The beads were incubated with HeLa nuclear extracts. Bound proteins were resolved by SDS PAGE, revealed by Coomassie staining and (E) analyzed on immunoblots with antibodies to CBF-A, hnRNP A2 and A3, and hnRNP U.

Article Snippet: Cloning, Expression, and Protein Purification Full-length hnRNP A2 (forward primer 5′-GGAATTCTTAGCGACTGAGTCCGCGATG, reverse primer 5′-ATAAGAATGCGGCCGCTGAAGCTGTTCTGTTACCTCTG) and hnRNP A3 ( Ma et al. , 2002 ) were cloned in pGEM-T (Promega, Madison, WI) and subsequently in pET30a (+) for expression (Novagen, Madison, WI).

Techniques: Affinity Purification, SDS Page, Staining, Western Blot, Recombinant, Construct, Incubation

Journal:

Article Title: In Cultured Oligodendrocytes the A/B-type hnRNP CBF-A Accompanies MBP mRNA Bound to mRNA Trafficking Sequences

doi: 10.1091/mbc.E07-10-1083

Figure Lengend Snippet: CBF-A binds the MBP mRNA RTS. (A) Sequences of wild-type (wtRTS) and scrambled RTS (scrRTS) used in this study. (B) Biotinylated wtRTS and scrRTS were conjugated to streptavidin Sepharose. Beads were incubated with HeLa nuclear, cytoplasmic, and high-salt protein extracts. Bound proteins were resolved by SDS-PAGE, revealed with Coomassie, and analyzed on immunoblots with antibodies to CBF-A and hnRNP A2 and A3. (C) RTS-binding assays using 33P-labeled wtRTS and scrRTS sequences. To perform EMSA, wtRTS and scrRTS probes were incubated with purified CBF-A and hnRNP A2 and A3 without affinity tags or (D) in the presence (+) or absence (−) of a 25-fold excess of unlabeled competitor RNA oligonucleotides as indicated. (E) Tissue distribution of CBF-A, analyzed on immunoblots, and normalized to the steady-state expression of histone H3.

Article Snippet: Cloning, Expression, and Protein Purification Full-length hnRNP A2 (forward primer 5′-GGAATTCTTAGCGACTGAGTCCGCGATG, reverse primer 5′-ATAAGAATGCGGCCGCTGAAGCTGTTCTGTTACCTCTG) and hnRNP A3 ( Ma et al. , 2002 ) were cloned in pGEM-T (Promega, Madison, WI) and subsequently in pET30a (+) for expression (Novagen, Madison, WI).

Techniques: Incubation, SDS Page, Western Blot, Binding Assay, Labeling, Purification, Expressing

Journal:

Article Title: In Cultured Oligodendrocytes the A/B-type hnRNP CBF-A Accompanies MBP mRNA Bound to mRNA Trafficking Sequences

doi: 10.1091/mbc.E07-10-1083

Figure Lengend Snippet: In cultured oligodendrocytes, CBF-A exhibits a granular cytoplasmic distribution which correlates with transported MBP mRNA. (A) Endogenous CBF-A (A–D and E–H) or (hnRNP A2 I–L and M–P) and MBP mRNA were simultaneously monitored by immuno-FISH and confocal microscopy. In D, arrows identify sites in which the distribution of CBF-A correlates with MBP RTS along processes. In E–H and M–P, oligodendrocyte processes are shown at approximately fivefold higher magnification. In H, arrowheads identify examples of CBF-A and MBP RTS-positive granules. In P, arrows point to examples of hnRNP A2- and MBP mRNA-positive granules. Scale bar, 20 μm. (B) Unbiased statistical quantification of individual CBF-A and MBP RTS-positive granules and (C) hnRNP A2 and MBP RTS-positive granules based on the immuno-FISH analysis. In both cases a linear correlation between the fluorescence intensity levels of CBF-A and RTS or hnRNP A2 and RTS is revealed.

Article Snippet: Cloning, Expression, and Protein Purification Full-length hnRNP A2 (forward primer 5′-GGAATTCTTAGCGACTGAGTCCGCGATG, reverse primer 5′-ATAAGAATGCGGCCGCTGAAGCTGTTCTGTTACCTCTG) and hnRNP A3 ( Ma et al. , 2002 ) were cloned in pGEM-T (Promega, Madison, WI) and subsequently in pET30a (+) for expression (Novagen, Madison, WI).

Techniques: Cell Culture, Confocal Microscopy, Fluorescence

Journal:

Article Title: In Cultured Oligodendrocytes the A/B-type hnRNP CBF-A Accompanies MBP mRNA Bound to mRNA Trafficking Sequences

doi: 10.1091/mbc.E07-10-1083

Figure Lengend Snippet: In oli-neu cells, the distribution of endogenous CBF-A correlates with hnRNP A2. (A and E) DAPI staining, (B and F) oli-neu cells stained with a mAb to hnRNP A2. (C and G) Oli-neu cells stained with the rabbit polyclonal peptide-specific anti-CBF-A antibody and (D and H) merged images. Scale bar, 20 μm. (B) Statistical quantification of CBF-A and hnRNP A2-positive granules based on the double immunofluorescence analysis and confocal microscopy in A. A linear correlation between the fluorescence signals of CBF-A and hnRNP A2 is revealed.

Article Snippet: Cloning, Expression, and Protein Purification Full-length hnRNP A2 (forward primer 5′-GGAATTCTTAGCGACTGAGTCCGCGATG, reverse primer 5′-ATAAGAATGCGGCCGCTGAAGCTGTTCTGTTACCTCTG) and hnRNP A3 ( Ma et al. , 2002 ) were cloned in pGEM-T (Promega, Madison, WI) and subsequently in pET30a (+) for expression (Novagen, Madison, WI).

Techniques: Staining, Immunofluorescence, Confocal Microscopy, Fluorescence

Journal:

Article Title: In Cultured Oligodendrocytes the A/B-type hnRNP CBF-A Accompanies MBP mRNA Bound to mRNA Trafficking Sequences

doi: 10.1091/mbc.E07-10-1083

Figure Lengend Snippet: CBF-A is associated with MBP mRNA in differentiating oligodendrocytes. (A) A complex containing CBF-A and hnRNP A2 is coprecipitated with the anti-CBF-A antibody from total protein extracts (Input) prepared from differentiating oli-neu cells in an RNA-dependent manner. Where indicated, extracts were treated with RNase A before immunoprecipitation. Bound proteins were resolved by SDS-PAGE and analyzed on immunoblots with antibodies to CBF-A and hnRNP A2. (B) qRT-PCR was performed on reverse-transcribed cDNA derived from RNA extracts of differentiating oli-neu cells, immunoprecipitated by CBF-A. The anti-CBF-A antibody leads to enrichment of MBP mRNA, as assessed with MBP-specific primers. Mock experiments and IgG pulldowns revealed negligible RNA enrichment. Input samples were considered to be 100%; thus all samples were divided by the inputs mean value. Data are presented as average of three independent experiments. Error bars, SEM. Importantly, in each case the percentages of immunoprecipitated mRNA are relative to the total amount of each individual mRNA species (input) analyzed.

Article Snippet: Cloning, Expression, and Protein Purification Full-length hnRNP A2 (forward primer 5′-GGAATTCTTAGCGACTGAGTCCGCGATG, reverse primer 5′-ATAAGAATGCGGCCGCTGAAGCTGTTCTGTTACCTCTG) and hnRNP A3 ( Ma et al. , 2002 ) were cloned in pGEM-T (Promega, Madison, WI) and subsequently in pET30a (+) for expression (Novagen, Madison, WI).

Techniques: Immunoprecipitation, SDS Page, Western Blot, Quantitative RT-PCR, Reverse Transcription, Derivative Assay

Journal: The Journal of Cell Biology

Article Title: The keratin-binding protein Albatross regulates polarization of epithelial cells

doi: 10.1083/jcb.200803133

Figure Lengend Snippet: Impaired AJC formation in Albatross knockdown cells. (A) Double staining for Albatross (red) and the undercoat proteins (green) for each AJC component: TJ, ZO-1; AJ, afadin; DS, desmoplakin. Top and bottom columns show projections of x-y planes and z sections, respectively. Albatross knockdown A549 (Albatross KD) cells lack accumulation of these proteins at the cell–cell borders except in regions where residual Albatross is present. (B) Cell–cell adhesive properties evaluated by a cell aggregation assay. In the differential interference contrast images, control cells show cell aggregation. With Albatross knockdown A549 (A1050 and A1160) cells, the aggregated cell population is reduced and free cells are increased. The percentages of single cells in total cells (mean ± SD) are: control, 36.1 ± 3.9; A1050, 52.4 ± 2.8; A1160 cells, 59.4 ± 10.2. n = 4 and P < 0.01. (C) Immunoelectron microscopy of A549 cells with anti-Albatross antibodies. Note that the cytoplasm in the vicinity of AJCs is labeled. TJ, AJ, and DS are indicated. Arrows indicate cell–cell contacts. (D) Quantitative data from C. (E) BC fraction and AJ fraction were immunostained for Albatross with the indicated AJC proteins, PKCζ or Par3. Note that Albatross is well colocalized with them. (F) Immunoblotting of fractions derived from mouse liver: homogenates (left), BC (middle), and AJ (right). Not only Albatross but also Par3 is enriched in line with the concentrations of the indicated AJC components. (G) Immunoprecipitation of A549 cells with anti-Albatross antibodies. Start and IP indicate starting lysates and immunoprecipitates with preimmune (Pre.) and anti-Albatross (αAlb.) antibodies, respectively. Note the Par3 precipitation with Albatross. Among AJC components, ZO-1 also coprecipitated. (H) Immunoprecipitation analysis with tagged Albatross and Par3. Start and IP indicate starting lysates and immunoprecipitates with anti-GFP antibodies, respectively. Left lanes show results for negative controls expressing GFP alone. Par3 was the most precipitated with GFP-Albatross among coexpressed myc-Par3, -Par6, and -PKCλ. Bars: (A) 10 μm; (B) 100 μm; (C) 0.1 μm; (E, BC) 13 μm; (E, AJ) 10 μm.

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-keratin 8 (Ks 8.7; Progen Pharmaceuticals), monoclonal mouse anti-keratin 18 (CY-90; Sigma-Aldrich), polyclonal mouse anti-pan keratin (Sigma-Aldrich), polyclonal guinea pig anti-K8/18 (Progen Pharmaceuticals), polyclonal guinea pig anti–desmoplakin 1 (Progen Pharmaceuticals), monoclonal mouse anti–desmoplakin 1 and 2 (Progen Pharmaceuticals), monoclonal mouse anti–ZO-1 (1; BD Biosciences), monoclonal rat anti–ZO-1 (BM173; Acris Antibodies, GmbH), monoclonal rat anti–E-cadherin (ECCD-2; EMD), monoclonal mouse anti-neurofilaments, monoclonal rat anti–platelet/endothelial cell adhesion molecule (anti-PECAM; CD31; BD Biosciences), monoclonal mouse anti–α-tubulin (B-5-1-2; Sigma-Aldrich), monoclonal mouse anti–claudin-2 (12H12; Invitrogen), monoclonal mouse anti–desmocollin-2/3 (7G6; Invitrogen), monoclonal mouse anti–desmoglein 2 (10G11; Progen Pharmaceuticals), monoclonal mouse anti–nectin-1 (CK8; Invitrogen), monoclonal mouse anti–β-catenin (14; BD Biosciences), polyclonal rabbit anti-ezrin (Millipore), rabbit anti-Par3 polyclonal antibody (provided by S. Ohno, Yokohama City University, Yokohama, Kanagawa, Japan; Millipore), monoclonal mouse anti-occludin (OC-3F10; Invitrogen), monoclonal rat anti–nectin-2 (502–57; HyCult Biotechnology), polyclonal rabbit anti-GFP (Santa Cruz Biotechnology, Inc.), polyclonal rabbit anti-PKCζ (Santa Cruz Biotechnology, Inc.), and polyclonal rabbit anti–glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) conjugated to HRP (Abcam).

Techniques: Double Staining, Immuno-Electron Microscopy, Labeling, Western Blot, Derivative Assay, Immunoprecipitation, Expressing

Journal: The Journal of Cell Biology

Article Title: The keratin-binding protein Albatross regulates polarization of epithelial cells

doi: 10.1083/jcb.200803133

Figure Lengend Snippet: Functions of keratins and Albatross–Par3 complexes. (A–C) The amounts of Albatross protein and mRNA were analyzed in both keratin 8 and keratin 18 (K8/18)-introduced SW13 cells. As a control, an empty vector was transfected. As loading controls, α-tubulin and GAPDH were used. Two independent experiments were performed. (A) Immunoblotting. In transiently K8/18-introduced SW13 cells, the amount of Albatross protein is elevated, along with the amount of keratin 18. (B) With stable lines, the same results were obtained. (C) RT-PCR. In K8/18-introduced SW13 cells, the mRNA level of K18 is elevated, but not that of Albatross. β-actin is included as an internal control. (D) Double staining for K8/18 and the indicated proteins: Albatross, AJC components of ZO-1 and afadin, and Par3. (top) In control cells, K8/18 is absent and only limited amounts of Albatross are apparent at cell–cell junctions. In stably K8/18-introduced SW13 cells, Albatross is well localized in cell–cell junctions compared with control cells. (middle and bottom) ZO-1, afadin, and Par3 similarly accumulated at the cell–cell borders in stably K8/18-introduced SW13 cells. (E) Immunostaining of stably K8/18-introduced SW13 cells transfected with control or Albatross siRNA. Note that ZO-1, afadin, and Par3 are reduced at cell–cell borders with knockdown of Albatross. (F) A model for the regulation of AJC and lateral domains with the Albatross–Par3 complex and keratins. Albatross–Par3 complexes regulate the formation of AJC and maintain lateral membrane identity. However, Par3 without Albatross regulates apical structures. Keratins stabilize Albatross, promoting the formation of AJC. Knockdown effects are also indicated. Bars, 10 μm.

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-keratin 8 (Ks 8.7; Progen Pharmaceuticals), monoclonal mouse anti-keratin 18 (CY-90; Sigma-Aldrich), polyclonal mouse anti-pan keratin (Sigma-Aldrich), polyclonal guinea pig anti-K8/18 (Progen Pharmaceuticals), polyclonal guinea pig anti–desmoplakin 1 (Progen Pharmaceuticals), monoclonal mouse anti–desmoplakin 1 and 2 (Progen Pharmaceuticals), monoclonal mouse anti–ZO-1 (1; BD Biosciences), monoclonal rat anti–ZO-1 (BM173; Acris Antibodies, GmbH), monoclonal rat anti–E-cadherin (ECCD-2; EMD), monoclonal mouse anti-neurofilaments, monoclonal rat anti–platelet/endothelial cell adhesion molecule (anti-PECAM; CD31; BD Biosciences), monoclonal mouse anti–α-tubulin (B-5-1-2; Sigma-Aldrich), monoclonal mouse anti–claudin-2 (12H12; Invitrogen), monoclonal mouse anti–desmocollin-2/3 (7G6; Invitrogen), monoclonal mouse anti–desmoglein 2 (10G11; Progen Pharmaceuticals), monoclonal mouse anti–nectin-1 (CK8; Invitrogen), monoclonal mouse anti–β-catenin (14; BD Biosciences), polyclonal rabbit anti-ezrin (Millipore), rabbit anti-Par3 polyclonal antibody (provided by S. Ohno, Yokohama City University, Yokohama, Kanagawa, Japan; Millipore), monoclonal mouse anti-occludin (OC-3F10; Invitrogen), monoclonal rat anti–nectin-2 (502–57; HyCult Biotechnology), polyclonal rabbit anti-GFP (Santa Cruz Biotechnology, Inc.), polyclonal rabbit anti-PKCζ (Santa Cruz Biotechnology, Inc.), and polyclonal rabbit anti–glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) conjugated to HRP (Abcam).

Techniques: Plasmid Preparation, Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Double Staining, Stable Transfection, Immunostaining