rabbit anti phospho tdp 43  (Proteintech)

Journal: Animal Cells and Systems

Article Title: Enhancer RNA-mediated transcriptional regulation of TDP-43 during early neural lineage specification

doi: 10.1080/19768354.2026.2643998

Figure Lengend Snippet: Regulatory roles of Tdp-43 eRNAs in pluripotency maintenance of mESCs (A) RT-qPCR analysis of Tdp-43 mRNA expression in mESCs following shRNA-mediated knockdown of either Tdp-43 or scrambled control. (B) Immunofluorescence analysis of TDP-43 protein expression in mESCs following shRNA-mediated knockdown of Tdp-43 compared to scrambled control (left). Quantification of fluorescence intensity (right) was performed using ImageJ and is presented as mean ± SEM from two independent biological replicates (n = 2). (C) Western blot analysis of TDP-43 (45 kDa) protein expression in mESCs following shRNA-mediated knockdown of Tdp-43 compared with scrambled control (left). α-Tubulin (55 kDa) was used as a loading control. Band intensities (right) were quantified using ImageJ and are presented as mean ± SEM from two independent biological replicates (n = 2). (D) RT-qPCR analysis of Tdp-43 eRNAs (E1-E4, sense strand) following knockdown of Tdp-43 or scrambled control. (E, F) RT-qPCR analysis of intergenic (E1-E3, E) and intragenic (E4, F) Tdp-43 eRNA (sense strand) following individual eRNA knockdown compared to scrambled control. (G) RT-qPCR analysis of Tdp-43 mRNA expression following knockdown of each eRNA compared to the scrambled control. (H) AP staining of mESCs following knockdown of scrambled control, Tdp-43 mRNA, intergenic eRNAs (E1-E3), or intragenic eRNA (E4, sense strand). (I, J) RT-qPCR analysis of pluripotency markers ( Pou5f1 , Sox2 , Nanog , and Klf4 ) and lineage-specific markers for ectoderm ( Nestin and Pax6 ), endoderm ( Sox17 ), and mesoderm ( T; Brachyury ) following knockdown of scrambled control and Tdp-43 mRNA (I), or scrambled control, intergenic eRNAs (E1-E3), and intragenic eRNA (E4) (J). The expression level of the intragenic eRNA was normalized by dividing the cDNA synthesized with random hexamers by that synthesized with oligo(dT) primers. All RT-qPCR data are presented as mean ± SEM from three independent biological replicates (n = 3). Statistical significance was assessed using an unpaired Student’s t-test. (K) Western blot analysis of TDP-43 (45 kDa) and NANOG (29-42 kDa) protein levels in mESCs following shRNA-mediated knockdown of the Tdp-43 intragenic eRNA (E4). α-Tubulin (55 kDa) was used as a loading control. Band intensities were quantified using ImageJ and are presented as mean ± SEM from two independent biological replicates (n = 2).

Article Snippet: Primary antibodies against TDP-43, NANOG and NESTIN (Cell Signaling Technology, Massachusetts; 89789S, 8822T and 10959, respectively) (1:1,000 in blocking buffer) were applied for 2 h at room temperature or overnight at 4°C, followed by three washes in TBS-T. HRP-conjugated secondary antibodies (1:5,000 in TBS-T) were then applied for 2 h at room temperature, followed by four washes.

Techniques: Quantitative RT-PCR, Expressing, shRNA, Knockdown, Control, Immunofluorescence, Fluorescence, Western Blot, Staining, Synthesized

Journal: Animal Cells and Systems

Article Title: Enhancer RNA-mediated transcriptional regulation of TDP-43 during early neural lineage specification

doi: 10.1080/19768354.2026.2643998

Figure Lengend Snippet: Dynamic expression of Tdp-43 eRNAs during ESC-to-NPC differentiation (A) Schematic representation of the ESC-to-NPC differentiation protocol used in this study. (B) RT-qPCR analysis of pluripotency markers ( Pou5f1 , Sox2 , Nanog , and Klf4 ) during the differentiation process from ESCs to NPCs. (C) RT-qPCR analysis of lineage-specific differentiation markers for ectoderm ( Nestin and Pax6 ), endoderm ( Sox17 ), and mesoderm ( T;brachyury ) during ESC-to-NPC differentiation. (D) Immunofluorescence analysis of NESTIN protein expression in mESCs and NPCs (left). Quantification of fluorescence intensity (right) was performed using ImageJ and is presented as mean ± SEM from two independent biological replicates (n = 2). (E) Western blot analysis of NESTIN (220 kDa) protein expression in ESCs and NPCs (left). α-Tubulin (55 kDa) was used as a loading control. Band intensities (right) were quantified using ImageJ and are presented as mean ± SEM from two independent biological replicates (n = 2). (F) RT-qPCR analysis of Tdp-43 mRNA expression in ESC and NPC states, shown as both bar graph (top) and line graph (bottom). RT-qPCR data are presented as mean ± SEM from four independent biological replicates (n = 4). Statistical significance was assessed using an unpaired Student’s t-test. (G, H) RT-qPCR analysis of intergenic sense eRNAs (E1-E3) (G) and the intragenic sense eRNA (E4) (H) transcribed from the Tdp-43 enhancer in ESC and NPC states, presented as bar graphs (top) and line graphs (bottom). The expression level of the intragenic eRNA was normalized by dividing the cDNA synthesized with random hexamers by that synthesized with oligo(dT) primers. All RT-qPCR data are presented as mean ± SEM from three independent biological replicates (n = 3), except for F. Statistical significance was assessed using an unpaired Student’s t-test.

Article Snippet: Primary antibodies against TDP-43, NANOG and NESTIN (Cell Signaling Technology, Massachusetts; 89789S, 8822T and 10959, respectively) (1:1,000 in blocking buffer) were applied for 2 h at room temperature or overnight at 4°C, followed by three washes in TBS-T. HRP-conjugated secondary antibodies (1:5,000 in TBS-T) were then applied for 2 h at room temperature, followed by four washes.

Techniques: Expressing, Quantitative RT-PCR, Immunofluorescence, Fluorescence, Western Blot, Control, Synthesized

Journal: Animal Cells and Systems

Article Title: Enhancer RNA-mediated transcriptional regulation of TDP-43 during early neural lineage specification

doi: 10.1080/19768354.2026.2643998

Figure Lengend Snippet: Functional requirement of retained eRNAs for Tdp-43 expression and NPC lineage specification (A) Schematic diagram depicting lentiviral shRNA infection during the differentiation of ESCs into NPCs. (B) RT-qPCR analysis of Tdp-43 mRNA expression in NPCs following shRNA-mediated knockdown of either Tdp-43 or scrambled control. (C-E) RT-qPCR analysis of Tdp-43 intergenic eRNAs (E1-E3, sense strand) (C), intragenic eRNA (E4, sense strand) (D), and Tdp-43 mRNA expression (E) in NPCs following individual eRNA knockdown, compared to scrambled control. (F) RT-qPCR analysis of lineage-specific markers for ectoderm (Nestin and Pax6 ) following knockdown of Tdp-43 mRNA, or intergenic (E2) and intragenic (E4) eRNAs, with the scrambled control included for comparison. (G) Schematic illustration of the WT eRNA construct design used for lentiviral rescue experiments. The diagram depicts the oligonucleotides used to generate the shRNA constructs for eRNA overexpression (WT eRNA_123 nt) and rescue experiments, along with the lentiviral vector backbone and shRNA expression cassette. (H, I) RT-qPCR analysis of Tdp-43 _E4 eRNA (H) and Tdp-43 mRNA (I) levels in ESCs following depletion of Tdp-43 _E4 eRNA or scrambled control. After knockdown, cells were analyzed with or without infection of WT E4 eRNA rescue lentivirus prior to RNA extraction. The expression level of the intragenic eRNA was normalized by dividing the cDNA synthesized with random hexamers by that synthesized with oligo(dT) primers. RT-qPCR data are presented as mean ± SEM from three independent biological replicates (n = 3). Statistical significance was assessed using an unpaired Student’s t-test. All RT-qPCR data are presented as mean ± SEM from four independent biological replicates (n = 4), except for H, I Statistical significance was assessed using an unpaired Student’s t-test.

Article Snippet: Primary antibodies against TDP-43, NANOG and NESTIN (Cell Signaling Technology, Massachusetts; 89789S, 8822T and 10959, respectively) (1:1,000 in blocking buffer) were applied for 2 h at room temperature or overnight at 4°C, followed by three washes in TBS-T. HRP-conjugated secondary antibodies (1:5,000 in TBS-T) were then applied for 2 h at room temperature, followed by four washes.

Techniques: Functional Assay, Expressing, shRNA, Infection, Quantitative RT-PCR, Knockdown, Control, Comparison, Construct, Over Expression, Plasmid Preparation, RNA Extraction, Synthesized

Journal: Scientific Reports

Article Title: Impact of G-quadruplex RNA oxidation on its conformational dynamics and interaction with ALS-associated TDP-43

doi: 10.1038/s41598-026-39767-y

Figure Lengend Snippet: Oxidative stress induced the conformational change of G4s. ( A ) Flowchart of the cellular oxidative stress experiment. Twelve hours after the addition of hydrogen peroxide, the GOTO neuroblastoma cells were fixed and immunostained. Then, total RNA was purified from the cells and used for the TO1B turn-on assay. ( B ) Representative immunofluorescence images showing TDP-43 and G4 signals detected using anti-TDP-43 and anti-G4 antibodies. Single-channel images and DAPI staining are shown, along with merged views. For quantification (bottom panels), the fluorescence intensities of TDP-43 and G4 were measured in ten cells per condition; values are normalized to untreated cells. Data represent mean ± SEM. Statistical significance was assessed using a two-tailed Student’s t-test (* P < 0.05). ( C ) Binding model of G4BPs. The proteins are classified into two groups: those that bind to prevent G4 formation (group 1) and those that stabilize G4 structures (group 2). ( D ) Measurement of G4 levels using the G4-specific fluorescent sensor TO1B. The fluorescence emission spectra of TO1B were measured after adding RNA extracted from untreated cells and cells oxidatively stressed with 20, 40, and 80 µM hydrogen peroxide. The G4-independent fluorescence intensity is also shown as a control. The graph on the right displays the average fluorescence intensity at 530 nm. The experiments were performed in triplicate, and the values on the y-axis represent the mean ± SEM. Statistical significance was determined using a two-tailed Student’s t -test. * P < 0.05.

Article Snippet: Immunofluorescence staining of cultured cells was performed as previously described , using a rabbit polyclonal anti-TDP-43 antibody (Cell Signaling Technology Inc., #3449 Danvers, Massachusetts, USA) and the mouse monoclonal anti-G-quadruplex antibody BG4 (Absolute Antibody Ltd., Ab00174-1.1 Redcar, UK).

Techniques: Purification, Immunofluorescence, Staining, Fluorescence, Two Tailed Test, Binding Assay, Control

Journal: Scientific Reports

Article Title: Impact of G-quadruplex RNA oxidation on its conformational dynamics and interaction with ALS-associated TDP-43

doi: 10.1038/s41598-026-39767-y

Figure Lengend Snippet: G4 oxidation reduces its interaction with TDP-43. ( A , B ) Agarose gel mobility shift assay. Fifty nanograms (0.5 pmol) of fluorescently labeled G4 probes ( Ax647 PSD-95-G4 or Ax647 CaMKIIα-G4) was mixed with two pmol of TDP-43 and the indicated unlabeled RNA competitors (threefold or 10-fold molar excess relative to the probe) and then electrophoresed under nondenaturing conditions. The shifted bands were quantified, and the mean and standard error of the mean (± SEM) were calculated from three independent experiments and displayed in the graph. The y-axis represents the mean ± SEM. Statistical significance was determined by two-tailed Student’s t -test. * P < 0.05, ** P < 0.01, *** P < 0.001. The red arrow indicates unexpected inhibition by the oxidized form of CaMKIIα-G4 (40% 8OG). These smear signals, indicated by dagger (†), have been confirmed to be the result of dissociation during electrophoresis . ( C ) CD spectra of PSD-95-G4 and oxidized RNAs (2 µM) in the absence and presence of TDP-43 (2 µM). The scans were repeated five times, and the protein spectra were subtracted; the mean values are shown. The CD spectrum of TDP-43 without RNA is also included. ( D ) Schematic illustration of G4 stabilization by TDP-43. The CD spectra of the oxidized RNA showed a decrease in the positive peak in the presence of TDP-43, indicating the stabilization of the transition intermediate G4 conformation.

Article Snippet: Immunofluorescence staining of cultured cells was performed as previously described , using a rabbit polyclonal anti-TDP-43 antibody (Cell Signaling Technology Inc., #3449 Danvers, Massachusetts, USA) and the mouse monoclonal anti-G-quadruplex antibody BG4 (Absolute Antibody Ltd., Ab00174-1.1 Redcar, UK).

Techniques: Agarose Gel Electrophoresis, Mobility Shift, Labeling, Two Tailed Test, Inhibition, Electrophoresis, Circular Dichroism

Journal: Scientific Reports

Article Title: Impact of G-quadruplex RNA oxidation on its conformational dynamics and interaction with ALS-associated TDP-43

doi: 10.1038/s41598-026-39767-y

Figure Lengend Snippet: Vulnerability of ALS mutant proteins to G4 oxidation. ( A ) TDP-43, which is encoded by the TARDBP gene, consists of two RRM with strong RNA-binding properties and a glycine-rich domain that regulates specific interactions. Ten mutations associated with patients with ALS, which were used in this study, are shown. The order/disorder propensity of TDP-43 was predicted using the Protein DisOrder prediction System (PrDOS) algorithm . ( B ) Purified TDP-43 wild-type and 10 mutant proteins used in this study. One microgram each of proteins was separated by 12.5% SDS-PAGE and detected by Coomassie brilliant blue staining. ( C ) Agarose gel mobility shift assay with Ax647 PSD-95-G4. Wild-type TDP-43 and the 10 mutant proteins were reacted with the probe in the presence or absence of competitor RNA (10% 8OG, 10-fold for probe). ( D ) Each overshifted band was quantified, and the mean and standard error (± SEM) obtained from three independent experiments were calculated and shown in the graph. E , Graph showing the average binding with and without competitor RNA. The ± SEM from three independent experiments is displayed. Statistical significance was assessed using a two-tailed Student’s t -test. * P < 0.05.

Article Snippet: Immunofluorescence staining of cultured cells was performed as previously described , using a rabbit polyclonal anti-TDP-43 antibody (Cell Signaling Technology Inc., #3449 Danvers, Massachusetts, USA) and the mouse monoclonal anti-G-quadruplex antibody BG4 (Absolute Antibody Ltd., Ab00174-1.1 Redcar, UK).

Techniques: Mutagenesis, RNA Binding Assay, Purification, SDS Page, Staining, Agarose Gel Electrophoresis, Mobility Shift, Binding Assay, Two Tailed Test