Mouse anti-hnRNP M3/4 Monoclonal Antibody, Purified [2A6-2H3] A500-011A from Bethyl Laboratories, Inc.

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Cell death & disease
October 03 2019

Fig 1: Overexpression of hnRNPM up-regulates NEAT1 expression and induces neurotoxicity.a NSC-34 cells were infected with adenovirus encoding FLAG-PR100 at MOIs of 0–800. To keep the constant total MOIs of adenoviruses, appropriate MOIs of LacZ-encoding adenovirus were added for each infection. At 48 h after the infection, the cell lysates were subjected to immunoblotting (IB) and dot blotting analysis using indicated antibodies. Intensities of immunodetected signals of endogenous hnRNPM were densitometrically examined with an ImageJ software. b, c NSC-34 cells were infected with adenovirus encoding FLAG-PR100 at MOIs of 0–800. To keep the constant total MOIs of adenoviruses, appropriate MOIs of LacZ-encoding adenovirus were added for each infection. At 48 h after the infection, the quantitative real-time PCR analysis of hnRNPM was performed (b). The cell lysates were subjected to dot blotting analysis using indicated antibodies (c). The data are presented as means ± SD (N = 3). Statistical analysis was performed by one-way ANOVA followed by the Dunnett’s multi comparisons test. d, e NSC-34 cells were infected with adenovirus encoding LacZ, hnRNPM, MATR3, or hnRNPQ at an MOI of 400. At 48 h after the infection, the quantitative real-time PCR analysis of NEAT1 was performed (d). The cell lysates were subjected to immunoblotting (IB) using indicated antibodies (e). The data are presented as means ± SD (N = 3). Statistical analysis was performed by one-way ANOVA followed by the Tukey’s multi comparisons test. f, g NSC-34 cells were transfected with 0.2 µg/well of the pCMV-GFP or -mouse NEAT1_2 on 6-well plate. After the transfection, NSC-34 cells were infected with adenovirus encoding LacZ or hnRNPM at an MOI of 400. At 48 h after the infection, the quantitative real-time PCR analysis of NEAT1 was performed (f). The cell lysates were subjected to immunoblotting (IB) using indicated antibodies (g). The data are presented as means ± SD (N = 3). Statistical analysis was performed by one-way ANOVA followed by the Dunnett’s multi comparisons test. h, i NSC-34 cells were infected with adenovirus encoding LacZ, hnRNPM, MATR3, or hnRNPQ at an MOI of 400. At 48 h after the infection, the cell viability was detected by WST-8 assay (h). The cell lysates were subjected to immunoblotting (IB) using indicated antibodies (i). The data are presented as means ± SD (N = 3). Statistical analysis was performed by one-way ANOVA followed by the Tukey’s multi comparisons test
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Cell death & disease
October 03 2019

Fig 2: Poly-proline-arginine (Poly-PR) associates with paraspeckle components.a Glutathione S-transferase (GST) or GST-FLAG-PR100 (GF-PR)-bound glutathione beads were mixed with ( + ) or without (-) NSC-34 cell lysates. After the rotation at 4 °C for 5 h, the glutathione beads were washed and were subjected to 5–20% gradient gel SDS–PAGE followed by immunoblotting (IB) using indicated antibodies. In western blotting using anti-GST antibody, the large smear within the molecular weights ranging 25–46 kDa in the GF-PR lane is thought to consist of C-terminal truncated GST-FLAG-PR100 proteins. A band located around 50 kDa in the GST lane is thought to represent dimerized and/or aggregated GST-derived proteins. b NSC-34 cells overexpressing EGFP-FLAG-PR100 (EGFP-F-PR100) (green) together with HA-tagged hnRNPF, hnRNPH1, or hnRNPM were fixed and were immunostained with HA (red). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, blue). Scale bar = 10 µm. The rightmost panel shows the profile image of fluorescence intensities on the lines of EGFP-FLAG-PR100 and hnRNPs-merged image. Arrows and arrowheads indicate the localization of nucleoplasm-localizing EGFP-FLAG-PR100 and the border of nucleolus-localizing EGFP-FLAG-PR100, respectively. c Lysates of NSC-34 cells overexpressing HA-tagged hnRNPF, hnRNPH1, or hnRNPM and GST or GST-FLAG-PR100 (GF-PR)-bound glutathione beads were incubated with ( + ) or without (-) 20 µg/mL RNase A. After the incubation, cell lysates were mixed with GST or GST-FLAG-PR100-bound glutathione beads. The glutathione beads were washed and were subjected to immunoblotting (IB) using indicated antibodies. d, e NSC-34 cells were transfected with the empty vector or the FLAG-PR100-encoding vector. At 48 h after the transfection, the cell lysates were immunoprecipitated (IP) with normal mouse IgG1 (Cont.) or the FLAG antibody. Precipitates were then used for RNA immunoprecipitation (RIP) assay (d) and dot blotting analysis with the FLAG antibody (e). Reverse transcription (RT) (-) was used as negative control to monitor the PCR amplification from genomic DNA. f NSC-34 cells overexpressing EGFP or EGFP-FLAG-PR100 (green) were fixed and stained with the 500 nM Quasar 570-labeled NEAT1 Stellaris probe (red). Nuclei were stained with DAPI (blue). Scale bar = 10 µm. Arrowheads and arrow correspond to paraspeckles. The rightmost panel shows the profile image of fluorescence intensities on the lines of EGFP-FLAG-PR100 and NEAT1-merged image in magnified image. Arrowhead and arrow indicate the co-localization of NEAT1 with nucleoplasm-localizing EGFP-FLAG-PR100 and the nucleolus-localizing EGFP-FLAG-PR100, respectively

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Mouse anti-hnRNP M3/4 Monoclonal Antibody, Purified [2A6-2H3]

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Bethyl Laboratories, Inc.'s Mouse anti-hnRNP M3/4 Monoclonal Antibody, Purified [2A6-2H3] is a Mouse monoclonal antibody. The Mouse anti-hnRNP M3/4 Monoclonal Antibody, Purified [2A6-2H3] was generated using HNRNPM as the antigen. It reacts with Homo Sapiens, Human, and Mouse. This antibody has been shown to work in applications such as: Precipitation, Immunoprecipitation, and Western Blot.

Cell death & disease

Cell death & disease

Mouse anti-hnRNP M3/4 Monoclonal Antibody, Purified [2A6-2H3] from Bethyl Laboratories, Inc.

Bethyl Laboratories, Inc.

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Mouse anti-hnRNP M3/4 Monoclonal Antibody, Purified [2A6-2H3] from Bethyl Laboratories, Inc.

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