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: Parasites & Vectors

Article Title: A mitochondrial HSP70 (HSPA9B) is linked to miltefosine resistance and stress response in Leishmania donovani

doi: 10.1186/s13071-016-1904-8

Figure Lengend Snippet: Effect of HSPA9B overexpression on differentiation. a L. donovani WT and transgenic promastigotes (HSPA9B-mCherry) were inoculated in low pH (pH = 5.5) medium and 37 °C to trigger differentiation to axenic amastigotes. Cells were collected 24 and 48 h post-induction of differentiation and either lysed for western blotting with axenic amastigote A2 marker and anti-tubulin as a loading control or counted using a Neubauer chamber. b The molecular weight of standard proteins is indicated in kDa and the mean ± standard deviation (SD) of triplicate determinations are shown, respectively

Article Snippet: Crude cell lysates, digitonin enriched and membrane and lumen organelle enriched fractions were separated in 4–12% Bis-Tris NuPAGE gels (Life, Carlsbad, USA) and revealed using the following antibodies: anti-GFP-horseradish-peroxidase (HRP)-conjugated antibody (Miltenyi, Bergisch Gladbach, Germany), rabbit polyclonal anti-mCherry antibody (Abcam, Cambridge, UK), mouse monoclonal anti-A2 antibody (Abcam, Cambridge, UK), mouse monoclonal anti-tubulin antibody (Sigma-Aldrich, St. Louis, USA), rabbit polyclonal anti-lipoic acid antibody (Abcam, Cambridge, UK) and anti-rabbit or anti-mouse HRP-conjugated secondary antibodies (Pierce, Waltham, USA).

Techniques: Over Expression, Transgenic Assay, Western Blot, Marker, Molecular Weight, Standard Deviation

Journal: Blood

Article Title: Reduced expression of annexin A2 is associated with impaired cell surface fibrinolysis and venous thromboembolism

doi: 10.1182/blood.2020008123

Figure Lengend Snippet: Characteristics of VTE subjects and HCs

Article Snippet: All antibodies were diluted in 5% nonfat milk in TBS-T. Primary antibodies included mouse monoclonal anti-human annexin A2 (1:1000; BD, #610069), rabbit monoclonal anti-human annexin A2 (1:1000; Cell Signaling, #D11G2), and anti-human annexin A2 (1:5000; kindly provided by Pfizer), as well as mouse monoclonal anti-human S100A10 (1:3000; BD Bioscience, #610070), mouse monoclonal anti-human glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:13 000; Meridian, #H86504M), and rabbit polyclonal anti-human annexin A5 (1:1000; Cell Signaling, #8555S).

Techniques:

Journal: Blood

Article Title: Reduced expression of annexin A2 is associated with impaired cell surface fibrinolysis and venous thromboembolism

doi: 10.1182/blood.2020008123

Figure Lengend Snippet: Cell surface tPA-dependent plasmin generation in VTE. (A) PBMC-related plasmin generating capacity. PBMCs from HCs and subjects with history of VTE were incubated with human Glu-plasminogen, washed, and then incubated with tPA (Genentech) in the presence of the fluorogenic plasmin substrate AFC-81. Shown are individual RFU/min2 values as well as means ± SEM for 60 HCs, 106 subjects with history of VTE, and VTE subgroups, including VTE with positive FH (VTE+FH, n = 45), VTE-FH (n = 61), VTE+FH+IT (n = 21), and VTE+FH-IT (n = 24). P values were determined using 1-way ANOVA and Tukey’s post hoc test of significance. Asterisks (* and **) indicate statistical significance (by Tukey’s post hoc vs unpaired t test, respectively). (B) Annexin A2–specific cell surface plasmin generation. HC PBMCs were incubated with Glu-plasminogen, followed by tPA (Molecular Innovations) and AFC-81 in the absence or presence of blocking monoclonal anti-A2 IgG or control IgG. Shown are values for mean RFU/min2 ± SEM from 3 separate experiments combined. The data were analyzed using 1-way ANOVA and Tukey’s post hoc test of significance. (C) Stratification of cell surface plasmin generation values. Values for cell surface plasmin generation (RFU/min2) depicted in panel A were stratified into 10th-percentile bins. The proportion of samples from each group falling into the lowest decile is indicated above each bar and by the black solid bars. NS, not significant; PN, plasmin.

Article Snippet: All antibodies were diluted in 5% nonfat milk in TBS-T. Primary antibodies included mouse monoclonal anti-human annexin A2 (1:1000; BD, #610069), rabbit monoclonal anti-human annexin A2 (1:1000; Cell Signaling, #D11G2), and anti-human annexin A2 (1:5000; kindly provided by Pfizer), as well as mouse monoclonal anti-human S100A10 (1:3000; BD Bioscience, #610070), mouse monoclonal anti-human glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:13 000; Meridian, #H86504M), and rabbit polyclonal anti-human annexin A5 (1:1000; Cell Signaling, #8555S).

Techniques: Incubation, Blocking Assay

Journal: Blood

Article Title: Reduced expression of annexin A2 is associated with impaired cell surface fibrinolysis and venous thromboembolism

doi: 10.1182/blood.2020008123

Figure Lengend Snippet: Effect of anticoagulation on plasmin generation, annexin A2 expression, and plasma CLT and CT. The data for cell surface plasmin generation (A), annexin A2 expression (B), plasma CLT (C), and plasma half-CT (D) were stratified according to anticoagulation therapy (AC) and compared with HCs. Shown are mean values ± SEM and P values derived by ANOVA and Tukey’s post hoc test of significance.

Article Snippet: All antibodies were diluted in 5% nonfat milk in TBS-T. Primary antibodies included mouse monoclonal anti-human annexin A2 (1:1000; BD, #610069), rabbit monoclonal anti-human annexin A2 (1:1000; Cell Signaling, #D11G2), and anti-human annexin A2 (1:5000; kindly provided by Pfizer), as well as mouse monoclonal anti-human S100A10 (1:3000; BD Bioscience, #610070), mouse monoclonal anti-human glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:13 000; Meridian, #H86504M), and rabbit polyclonal anti-human annexin A5 (1:1000; Cell Signaling, #8555S).

Techniques: Expressing, Derivative Assay

Journal: Blood

Article Title: Reduced expression of annexin A2 is associated with impaired cell surface fibrinolysis and venous thromboembolism

doi: 10.1182/blood.2020008123

Figure Lengend Snippet: Annexin A2 protein expression in VTE. (A) A2 protein expression in VTE subgroups. Annexin A2 to GAPDH expression ratios are indicated for 19 HCs, 41 subjects with history of VTE, 16 subjects with VTE+FH, 25 subjects with VTE−FH, 5 subjects with VTE+FH+IT, and 11 subjects with VTE+FH−IT. Shown are individual values, mean ± SEM, and P values determined using 1-way ANOVA and Tukey’s post hoc test of significance. Asterisks (* and **) indicate statistical significance (by Tukey’s post hoc vs unpaired t test, respectively). (B) Stratification of annexin A2 expression values into decile bins. The percent of subjects with A2/GAPDH ratios in the lowest 10th-percentile for each group are depicted by black bars and numbers above each bar.

Article Snippet: All antibodies were diluted in 5% nonfat milk in TBS-T. Primary antibodies included mouse monoclonal anti-human annexin A2 (1:1000; BD, #610069), rabbit monoclonal anti-human annexin A2 (1:1000; Cell Signaling, #D11G2), and anti-human annexin A2 (1:5000; kindly provided by Pfizer), as well as mouse monoclonal anti-human S100A10 (1:3000; BD Bioscience, #610070), mouse monoclonal anti-human glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:13 000; Meridian, #H86504M), and rabbit polyclonal anti-human annexin A5 (1:1000; Cell Signaling, #8555S).

Techniques: Expressing

Journal: Blood

Article Title: Reduced expression of annexin A2 is associated with impaired cell surface fibrinolysis and venous thromboembolism

doi: 10.1182/blood.2020008123

Figure Lengend Snippet: Epitope-specific immunoblotting. (A-B) Representative immunoblots depicting annexin A2, S100A10, annexin A5, and GAPDH expression in PBMCs. PBMC lysates from HCs as well as subjects with VTE and low (labeled in red) or normal A2 expression were probed with IgG directed against the core domain of A2, as well as IgGs directed against S100A10, GAPDH, and annexin A5. A sample from the same internal control (IC) was also examined in each blot. FH status, presence or absence of an IT, and presence or absence of the 5-SNP cluster are also indicated. Black dots indicate not tested. All results shown are representative of blots on samples collected on ≥2 separate occasions ≥3 months apart. L, low; N, normal.

Article Snippet: All antibodies were diluted in 5% nonfat milk in TBS-T. Primary antibodies included mouse monoclonal anti-human annexin A2 (1:1000; BD, #610069), rabbit monoclonal anti-human annexin A2 (1:1000; Cell Signaling, #D11G2), and anti-human annexin A2 (1:5000; kindly provided by Pfizer), as well as mouse monoclonal anti-human S100A10 (1:3000; BD Bioscience, #610070), mouse monoclonal anti-human glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:13 000; Meridian, #H86504M), and rabbit polyclonal anti-human annexin A5 (1:1000; Cell Signaling, #8555S).

Techniques: Western Blot, Expressing, Labeling

Journal: Blood

Article Title: Reduced expression of annexin A2 is associated with impaired cell surface fibrinolysis and venous thromboembolism

doi: 10.1182/blood.2020008123

Figure Lengend Snippet: Relative annexin A2 mRNA levels. mRNA was isolated from PBMCs as described in “Methods” and analyzed by quantitative RT-PCR. (A) mRNA levels were stratified according to normal (n = 6) or low A2 protein expression (n = 5), defined as below the 10th percentile for all subjects, and compared with mRNA levels in HCs (n = 6). All samples were run in triplicate. (B) mRNA levels were stratified according to the presence or absence of the 5-SNP cluster. Shown in panels A and B are mean relative mRNA levels ± SEM.

Article Snippet: All antibodies were diluted in 5% nonfat milk in TBS-T. Primary antibodies included mouse monoclonal anti-human annexin A2 (1:1000; BD, #610069), rabbit monoclonal anti-human annexin A2 (1:1000; Cell Signaling, #D11G2), and anti-human annexin A2 (1:5000; kindly provided by Pfizer), as well as mouse monoclonal anti-human S100A10 (1:3000; BD Bioscience, #610070), mouse monoclonal anti-human glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:13 000; Meridian, #H86504M), and rabbit polyclonal anti-human annexin A5 (1:1000; Cell Signaling, #8555S).

Techniques: Isolation, Quantitative RT-PCR, Expressing