Fig 1: 3′ UTR length variation during human motor neurogenesis. a Boxplots of the distributions of maximum 3′ UTR lengths expressed at distinct stages of MN differentiation in control (left) and VCPmu samples (right). P-value obtained with Wilcoxon test. b Bar plots displaying the numbers of 3′ UTR with statistically significant promoter-distal (left) and promoter-proximal (right) shifts at distinct stage of differentiation compared with iPSCs in control (grey bars) and VCPmu samples (magenta bars). Inset, pie charts representing the proportions of genes exhibiting alternative 3′ UTR usage in control and VCPmu samples (white), control samples only (grey area) or VCPmu samples only (magenta area). c GO enrichment analysis of biological pathways associated with genes showing statistically significant distal shifts in poly(A) site usage in control mMNs compared to control iPSCs. d, e Left, genome browser views of RNA-seq profiles in the 3′ UTRs of genes GNL1 and TARDBP which exhibit statistically significant proximal-to-distal shifts in poly(A) site usage in mMNs compared with iPSCs. Right, bar plots showing distal 3′ UTR usage relative to the proximal 3′ UTR. P-values obtained with Fisher count test. f Same as c for genes showing statistically significant proximal shifts in poly(A) site usage in control NPCs compared with control iPSCs. g, h Same as d for genes ZNF254 and DARS exhibiting statistically significant distal-to-proximal shifts in poly(A) site usage in NPCs compared with iPSCs
Fig 2: TDP-43 antibody screening by immunofluorescence.HAP1 WT and TARDBP KO cells were labelled with a green or a far-red fluorescent dye, respectively. WT and KO cells were mixed and plated to a 1:1 ratio in a 96-well plate with a glass bottom. Cells were stained with the indicated TDP-43 antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (identification of WT cells), red (antibody staining) and far-red (identification of KO cells) channels was performed. Representative images of the merged blue and red (grayscale) channels are shown. WT and KO cells are outlined on both channels with green and magenta dashed lines, respectively. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Exceptions were given for antibodies 12892-1-AP, MA5-32627**, A19123**, 89789**, 89718** and ab133547** which were titrated to 1/400, 1/1000, 1/900, 1/30, 1/10 and 1/700, respectively, as the signals were too weak when following the supplier’s recommendations When the concentration was not indicated by the supplier, which was the case for antibodies 80001-1-RR**, GTX630196* and ab254166**, we tested antibodies at 1/700, 1/1000 and 1/500, respectively. At these concentrations, the signal from each antibody was in the range of detection of the microscope used. Antibody dilution used: 10782-2-AP at 1/400, 12892-1-AP at 1/250, 800001-1-RR** at 1/700, 80002-1-RR** at 1/250, MAB7778* at 1/500, NBP1-92695* at 1/1000, 711051** at 1/500, MA5-27828* at 1/1000, MA5-32627** at 1/1000, A19123** at 1/900, 89789** at 1/30, 89718** at 1/10, GTX630196* at 1/1000, GTX630197* at 1/1000, ab109535** at 1/30, ab133547** at 1/700, ab190963** at 1/800, ab254166** at 1/500. Bars = 10 μm. *Monoclonal antibody, **Recombinant antibody.
Fig 3: TDP-43 antibody screening by Western blot.Lysates of HAP1 (WT and TARDBP KO) were prepared and 50 μg of protein were processed for Western blot with the indicated TDP-43 antibodies. The Ponceau stained transfers of each blot are shown. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Exceptions were given for antibody 80001-1-RR**, which was titrated to 1/1000 as the signal was too weak when following the supplier’s recommendations. When the concentration was not indicated by the supplier, which was the case for antibody 80002-1-RR**, we tested the antibody at 1/1000. Antibody dilution used: 10782-2-AP at 1/5000, 12892-1-AP at 1/1000, 800001-1-RR** at 1/1000, 80002-1-RR** at 1/1000, MAB7778* at 1/500, NBP1-92695* at 1/1000, 711051** at 1/1000, MA5-27828* at 1/1000, MA5-32627** at 1/1000, A19123** at 1/1000, 89789** at 1/1000, 89718** at 1/1000, GTX630196* at 1/500, GTX630197* at 1/500, ab109535** at 1/2000, ab133547** at 1/1000, ab190963** at 1/1000, ab254166** at 1/1000. Predicted band size: 45 kDa. *Monoclonal antibody, **Recombinant antibody.
Fig 4: TDP-43 antibody screening by immunoprecipitation.HAP1 lysates were prepared, and IP was performed using 2.0 μg of the indicated TDP-43 antibodies pre-coupled to Dynabeads protein G or protein A. Samples were washed and processed for Western blot with the indicated TDP-43 antibody. For Western blot, 80002-1-RR** was used at 1/1000. The Ponceau stained transfers of each blot are shown for similar reasons as in Figure 1. SM=4% starting material; UB=4% unbound fraction; IP=immunoprecipitate. *Monoclonal antibody, **Recombinant antibody.
Supplier Page from Abcam for Anti-TDP43 antibody [EPR5811]