buffer rabbit anti tdp43 antibody  (Proteintech)

Journal: Molecular cell

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia.

doi: 10.1016/j.molcel.2022.12.019

Figure Lengend Snippet: Figure 1. TDP43 recognizes m6A-modified RNA (A and B) UG density 100 bp up- and downstream of m6A modifications identified by cross-linking induced mutation sites (CIMSs) (A) or cross-linking induced truncation sites (CITSs) (B) in relation to random sequences (red line). Gray shading, 95% CI. (C) Schematic of HaloTag immunoprecipitation and dot blot procedure. (D) Dot blot for total RNA (detected by methylene blue) or m6A-modified RNA (detected by anti-m6A antibody) following HaloTag immunoaffinity purification in HEK293T cells overexpressing HaloTag, TDP43-HaloTag or YTHDF2-HaloTag from 3 biological replicates. (E) Diagram illustrating insertion of HaloTag downstream of the TARDBP start codon (green line), encoding HaloTag-TDP43. (F) Halo-TDP43 HEK293T cells labeled live with JF646 dye (red), then fixed, permeabilized, and immunostained for TDP43 (green) prior to imaging. DAPI (blue) marks the nucleus. Scale bars, 10 mm. (G) Dot blot for total and m6A RNA isolated by immunoaffinity purification of endogenous HaloTag-TDP43 or overexpressed HaloTag. Empty space between lanes was clipped in (D) and (E). Additional replicates shown in Figure S1.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Mutagenesis, Immunoprecipitation, Dot Blot, Labeling, Imaging, Isolation

Journal: Molecular cell

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia.

doi: 10.1016/j.molcel.2022.12.019

Figure Lengend Snippet: Figure 3. m6A modifications influence TDP43 binding and autoregulation (A) TARDBP gene map, illustrating TDP43 binding region (TBR), the location of the DRACH motif (pink square), and the C-T transition (red box) identified by DART- seq within this domain, representing an m6A site.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Binding Assay

Journal: Molecular cell

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia.

doi: 10.1016/j.molcel.2022.12.019

Figure Lengend Snippet: Figure 6. YTHDF2 reduction extends neuronal survival in human neuron disease models (A) YTHDF2 immunostaining in control and sALS spinal cord. Scale bars, 50 mm. (B) YTHDF2 immunoreactivity in spinal neurons from control (n = 117 neurons) and sALS (n = 193 neurons) samples. Plot shows mean ± SD, color coded by sample. ****p < 0.0001 via Mann-Whitney test. (C) Strategy used to create isogenic iPSCs expressing native TDP43(WT)-Dendra2 or TDP43(M337V)-Dendra2. (D–F) (D) Representative images of untransduced (gray) and transduced (green) iNeurons expressing shRNA against YTHDF2 (shYTHDF2). Time of death (red circles) for each cell is used to determine cumulative risk of death, plotted in (E) and (F). Scale bars, 20 mm. YTHDF2 knockdown significantly extended the survival of TDP43(M337V)-Dendra2 iNeurons (E; yp = 8.42 3 1012, HR = 6.25; ***p = 4.82 3 109, HR = 0.32; #p = 0.08, HR = 1.84) as well as mutant C9ORF72 iNeurons (F, yp = 1.42 3 1011, HR = 2.85; ***p = 1.42 3 1016, HR = 0.32). ns, not significant. Values in (E) and (F) calculated by Cox proportional hazards analysis, with a minimum 3 biological replicates.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Immunostaining, Control, MANN-WHITNEY, Expressing, shRNA, Knockdown, Mutagenesis