Journal: Brain research

Article Title: Rodent models of TDP-43: Recent advances

doi: 10.1016/j.brainres.2012.04.031

Figure Lengend Snippet: Rodent models of wild type or ALS-linked mutant TDP-43.

Article Snippet: TDP-43 fragments [antibody used] , 25 and 35 kDa fragments (detergent-soluble) in 1–2 months and older mice [Proteintech 10782-2-AP, anti-body to TDP-43N-260aa] , , Some low molecular weight fragments in spinal cord homogenate and cytosolic fractions [Proteintech 10782-2-AP, antibody to TDP-43N-260aa] , Low molecular weight fragments in spinal cord homogenate and cytosolic fractions [Proteintech 10782-2-AP, antibody to TDP-43N-260aa] , 25 and 35 kDa fragments in brain and spinal cord extract from hemizygous and homozygous mice [Proteintech 12892-1-AP, antibody to TDP-43 260aa-C] , 25 and 35 kDa fragments in brain lysates from nontransgenic, hemizygous, and homozygous mice [Proteintech 12892-1-AP, antibody to TDP-43 260aa-C].

Techniques: Mutagenesis, Expressing, Staining, Molecular Weight

Journal: Diagnostics

Article Title: CSF Diagnostics: A Potentially Valuable Tool in Neurodegenerative and Inflammatory Disorders Involving Motor Neurons: A Review

doi: 10.3390/diagnostics11091522

Figure Lengend Snippet: Information on cohort selection size and composition, body fluid, as well as used technique/antibody and detected TDP-43 levels among the assessed studies. Abbreviations: ALS = amyotrophic lateral sclerosis, FTLD = frontotemporal lobar degeneration, GBS = Guillain Barré syndrome, MS= multiple sclerosis, PD = Parkinson’s disease.

Article Snippet: Kasai et al., 2009 , 30 ALS, 29 controls (13 controls, 16 disease controls) , CSF , TDP-43 , Sandwich ELISA (Nunc MaxiSorp, Xat-bottom 96-well Black MicroWell plate, Roskilde, Denmark) , anti-TDP-43 monoclonal antibody, detection antibody, anti-TDP-43 rabbit polyclonal antibody (10782-2-AP, ProteinTech Group, Chicago, IL, USA), raised against a recombinant protein corresponding to residues 1–261 of human TDP-43 (H00023435-M01, clone 2E2-D3, Abnova Corporation, Walnut, CA, USA), detection antibody, anti-TDP-43 rabbit polyclonal antibody (10782-2-AP, ProteinTech Group, Chicago, IL, USA) , ALS: 6.92 +/− 3.71; Control: 5.31 +/− 0.94.

Techniques: Selection, Marker, Sandwich ELISA, Recombinant, Western Blot, Affinity Purification

Journal: Autophagy

Article Title: Cryptic exon splicing function of TARDBP interacts with autophagy in nervous tissue

doi: 10.1080/15548627.2018.1474311

Figure Lengend Snippet: Antibodies and conditions employed.

Article Snippet: Target Dilution Source TARDBP 1:1000 in TBS-T 0.05% Proteintech, 10782–2-AP ATG4B 1:250 in TBS-T 0.05% Sigma-Aldrich, A2981 SQSTM1 1:1000 in TBS-T 0.05% (Western blot); 1:100 in PBS (immunocytofluorescence) Cell Signalling Technology, 5114 Secondary anti-rabbit, HRP conjugate 1:50000 in TBS-T 0.05% Thermo Fisher Scientific, 31460 Secondary anti-rabbit, Alexa Fluor® 546 conjugate 1:800 in PBS Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A11010","term_id":"492391","term_text":"A11010"}} A11010 Open in a separate window Antibodies and conditions employed.

Techniques: Western Blot

Journal: bioRxiv

Article Title: Riluzole does not ameliorate disease caused by cytoplasmic TDP-43 in a mouse model of amyotrophic lateral sclerosis

doi: 10.1101/749846

Figure Lengend Snippet: A Representative immunoblots of RIPA-soluble and urea-soluble cortical brain lysates from vehicle- or riluzole-treated control and rNLS mice for human-specific (h)TDP-43, human- and mouse-specific (h+m)TDP-43 and phosphorylated (p409/410) TDP-43. Quantification of B RIPA-soluble hTDP-43ΔNLS, C RIPA-soluble h+mTDP-43, D urea-soluble hTDP-43ΔNLS, E urea-soluble h+mTDP-43, and F p409/410 TDP-43 revealed higher levels in rNLS mice, but no effect of riluzole treatment. GAPDH for loading control of hTDP-43 and h+mTDP-43 blot is shown, with full blots and individual matched GAPDH/total protein blots used for quantification shown in . n =3/control group and n =5/rNLS group. Detailed statistical test results are described in .

Article Snippet: Primary antibodies included: rabbit anti-human/mouse TDP-43 1:5000 (Proteintech Cat# 10782-2-AP, RRID:AB_615042), mouse anti-human TDP-43 1:5000 (Clone 5104, ( )), mouse anti-phospho-TDP-43 (Ser409/410) (Cosmo Bio Co Cat# CAC-TIP-PTD-M01, RRID:AB_1961900), rabbit anti-EAAT2 1:1000 (Abcam Cat# ab178401), rabbit anti-GluA1 1:1000 (Abcam Cat# ab31232, RRID:AB_2113447), rabbit anti-GluA2 1:1000 (Millipore Cat# AB1768, RRID:AB_2247874), rabbit anti-GluA3 1:1000 (Abcam Cat# ab40845, RRID:AB_776310), and mouse anti-GAPDH 1:5000 (Proteintech Cat# 60004-1-Ig, RRID:AB_2107436), diluted in 0.1% BSA overnight at 4°C.

Techniques: Western Blot

Journal: Acta Neuropathologica

Article Title: Low molecular weight species of TDP-43 generated by abnormal splicing form inclusions in amyotrophic lateral sclerosis and result in motor neuron death

doi: 10.1007/s00401-015-1412-5

Figure Lengend Snippet: TDP-43 r.[106_196del] splice variant is upregulated in ALS. a TARDBP gene structure showing splicing deletion. Black boxes are exons. White box in exon 2 denotes the 91 bp skipped by alternative splicing. Arrows indicate positions of the forward and reverse primers for RT-PCR. b RT-PCR amplification of the TDP-43 r.[106_196del] splice variant using RNA isolated from ALS and control spinal cord. Region amplified has 511 bp in the constitutively spliced transcript and 420 bp in the alternatively spliced transcript. Cases A1 , A4 , A5 , A9 , and A11 also carry C9orf72 expansions. c Quantification using ImageJ showed a ~4 fold upregulation of the splice variant in ALS ( n = 12; 0.216 ± 0.031) compared to controls ( n = 4; 0.059 ± 0.013), p < 0.0126

Article Snippet: Equivalent amounts of protein samples were loaded onto 10 % SDS-PAGE gels and transferred to polyvinyldiflouride (PVDF) membranes, which were blocked in 5 % skim milk dissolved in Tris-buffered saline (TBS) containing 0.2 % Tween-20 for 1 h at RT, followed by overnight incubation at 4 °C with a polyclonal TDP-43 antibody (1:1000, Proteintech), a polyclonal N-terminal TDP-43 antibody (1:1000, Aviva Systems Biology), or a polyclonal C-terminal TDP-43 antibody (1:1000, generated in-house using a synthetic peptide, FGSSMDSKSSGWG, linked to keyhole limpet hemocyanin as the antigen).

Techniques: Variant Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Isolation

Journal: Acta Neuropathologica

Article Title: Low molecular weight species of TDP-43 generated by abnormal splicing form inclusions in amyotrophic lateral sclerosis and result in motor neuron death

doi: 10.1007/s00401-015-1412-5

Figure Lengend Snippet: Sequence alignment of TDP-43 and the TDP-43 r.[106_196del] splice variant. The 91 bp splicing deletion is signified by dotted lines . Pat7 (aa 78–84, shown in bold italics ) and bipartite (aa 82–98, boxed ) nuclear localization sequences are indicated. Note that ATG Met85 immediately precedes the caspase-3 cleavage site DETD 89 ( underlined )

Article Snippet: Equivalent amounts of protein samples were loaded onto 10 % SDS-PAGE gels and transferred to polyvinyldiflouride (PVDF) membranes, which were blocked in 5 % skim milk dissolved in Tris-buffered saline (TBS) containing 0.2 % Tween-20 for 1 h at RT, followed by overnight incubation at 4 °C with a polyclonal TDP-43 antibody (1:1000, Proteintech), a polyclonal N-terminal TDP-43 antibody (1:1000, Aviva Systems Biology), or a polyclonal C-terminal TDP-43 antibody (1:1000, generated in-house using a synthetic peptide, FGSSMDSKSSGWG, linked to keyhole limpet hemocyanin as the antigen).

Techniques: Sequencing, Variant Assay

Journal: Acta Neuropathologica

Article Title: Low molecular weight species of TDP-43 generated by abnormal splicing form inclusions in amyotrophic lateral sclerosis and result in motor neuron death

doi: 10.1007/s00401-015-1412-5

Figure Lengend Snippet: TDP-43 r.[106_196del] splice variant generates an N-terminally truncated protein of 35 kDa. SH-SY5Y cells expressing full-length TDP-43, TDP-43 r.[106_196del], Met 85 -TDP-35, or mock vector were fractionated in buffers of increasing stringency: a low salt; b high salt-TX-100; c – e urea buffer. Full-length TDP-43 was found in all fractions on immunoblots probed with polyclonal TDP-43. A lower molecular weight species of 35 kDa was observed only in urea-soluble fractions of lysates from cells expressing TDP-43 r.[106_196del] or Met 85 -TDP-35 ( c ) and was detected using antibody to the C-terminus of TDP-43 ( d ) but not to the N-terminus ( e ). f Comparative structures of TDP-43 and Met 85 -TDP-35

Article Snippet: Equivalent amounts of protein samples were loaded onto 10 % SDS-PAGE gels and transferred to polyvinyldiflouride (PVDF) membranes, which were blocked in 5 % skim milk dissolved in Tris-buffered saline (TBS) containing 0.2 % Tween-20 for 1 h at RT, followed by overnight incubation at 4 °C with a polyclonal TDP-43 antibody (1:1000, Proteintech), a polyclonal N-terminal TDP-43 antibody (1:1000, Aviva Systems Biology), or a polyclonal C-terminal TDP-43 antibody (1:1000, generated in-house using a synthetic peptide, FGSSMDSKSSGWG, linked to keyhole limpet hemocyanin as the antigen).

Techniques: Variant Assay, Expressing, Plasmid Preparation, Western Blot, Molecular Weight

Journal: Acta Neuropathologica

Article Title: Low molecular weight species of TDP-43 generated by abnormal splicing form inclusions in amyotrophic lateral sclerosis and result in motor neuron death

doi: 10.1007/s00401-015-1412-5

Figure Lengend Snippet: Expression of Met 85 -TDP-35 causes death of primary motor neurons. a Expression of EGFP-tagged TDP-43 and EGFP-tagged Met 85 -TDP-35 in primary motor neurons resulted in localization of TDP-43 to the nucleus and Met 85 -TDP-35 to the cytoplasm, where it formed aggregates ( arrow ). b A viability curve showed that Met 85 -TDP-35 induced motor neuron death compared to TDP-43 or empty vector alone. Scale bar 20 μm

Article Snippet: Equivalent amounts of protein samples were loaded onto 10 % SDS-PAGE gels and transferred to polyvinyldiflouride (PVDF) membranes, which were blocked in 5 % skim milk dissolved in Tris-buffered saline (TBS) containing 0.2 % Tween-20 for 1 h at RT, followed by overnight incubation at 4 °C with a polyclonal TDP-43 antibody (1:1000, Proteintech), a polyclonal N-terminal TDP-43 antibody (1:1000, Aviva Systems Biology), or a polyclonal C-terminal TDP-43 antibody (1:1000, generated in-house using a synthetic peptide, FGSSMDSKSSGWG, linked to keyhole limpet hemocyanin as the antigen).

Techniques: Expressing, Plasmid Preparation

Journal: Acta Neuropathologica

Article Title: Low molecular weight species of TDP-43 generated by abnormal splicing form inclusions in amyotrophic lateral sclerosis and result in motor neuron death

doi: 10.1007/s00401-015-1412-5

Figure Lengend Snippet: Antibody to Met 85 -TDP-35 detects Met 85 -TDP-35 in ALS and ALS-FTLD tissues. a Synthetic peptide sequences used for generation of neo-epitope rabbit polyclonal antibodies specific to Met 85 -TDP-35 ( underlined in red ) and C3-TDP-35 ( underlined in green ). b – d Neo-epitope antibodies were characterized using lysates of cells expressing TDP-43 r.[106_196del], recombinant YFP-tagged full-length TDP-43 (YFP-TDP-43), and recombinant YFP-tagged full-length TDP-43 cleaved with caspase 3 (C3-cleaved). Note that there is a caspase-3 cleavage site at DEND 13 of full-length TDP-43, which cleaves the YFP tag ( asterisk ); and at DVMD 19 , generating a cleavage product of 25 kDa ( arrowhead ). e – g Urea-soluble fractions of spinal cord extracts from four ALS cases ( A1 – A4 ) and four controls ( C1 – C4 ) were immunoblotted with polyclonal TDP-43 antibody ( e ), Met 85 -TDP-35 antibody ( g ), and C3-TDP-35 antibody ( g ). The 35-kDa TDP-43 species ( arrowhead ) in ALS tissue was detected with polyclonal TDP-43 antibody and Met 85 -TDP-35 antibody but not with C3-TDP-35 antibody. Full-length TDP-43 ( arrow ) was not detected by the Met 85 -TDP-35 antibody. The location of the 25-kDa species is shown with an asterisk . The 72-kDa band detected by the C3-TDP-35 antibody is non-specific ( double asterisk ). h Urea-soluble fractions of prefrontal cortex extracts from six patients with ALS-FTLD and six controls were immunoblotted with Met 85 -TDP-35 antibody. In four out of six patient samples, a 35-kDa species corresponding to Met 85 -TDP-35 was apparent and was absent from all control samples. An additional band of 37 kDa was also apparent in one patient sample, the identity of which is unknown. Cases A1 , A4 , A5 , A9 , and A11 also carry C9orf72 expansions

Article Snippet: Equivalent amounts of protein samples were loaded onto 10 % SDS-PAGE gels and transferred to polyvinyldiflouride (PVDF) membranes, which were blocked in 5 % skim milk dissolved in Tris-buffered saline (TBS) containing 0.2 % Tween-20 for 1 h at RT, followed by overnight incubation at 4 °C with a polyclonal TDP-43 antibody (1:1000, Proteintech), a polyclonal N-terminal TDP-43 antibody (1:1000, Aviva Systems Biology), or a polyclonal C-terminal TDP-43 antibody (1:1000, generated in-house using a synthetic peptide, FGSSMDSKSSGWG, linked to keyhole limpet hemocyanin as the antigen).

Techniques: Expressing, Recombinant

Journal: Acta Neuropathologica

Article Title: Low molecular weight species of TDP-43 generated by abnormal splicing form inclusions in amyotrophic lateral sclerosis and result in motor neuron death

doi: 10.1007/s00401-015-1412-5

Figure Lengend Snippet: Immunohistochemical characterization of Met 85 -TDP-35 in ALS spinal cord. a Immunohistochemical analysis of serial sections from ALS and labeled with polyclonal TDP-43 antibody (poly-TDP-43 Ab) and Met 85 -TDP-35 antibody (Met 85 -TDP-35 Ab). Note that Met 85 -TDP-35 immunoreactivity is localized to TDP-43-positive pathology ( arrows ). b Met85-TDP-35 immunoreactivity was ablated by competition with the immunizing peptide ( arrows ). c There was no coincident labeling of TDP-43 pathology with C3-TDP-35 antibody (C3-TDP-35 Ab). d Double immunofluorescence labeling of control and ALS spinal motor neurons with mouse monoclonal TDP-43 antibody (Mono TDP-43; red ) and Met 85 -TDP-35 Ab ( green ), nuclei stained with DAPI ( blue ). Note labeling of motor neuron nucleus in control spinal cord with mono TDP-43 antibody but not with Met 85 -TDP-35 antibody ( arrowhead ), indicating lack of cross reactivity of Met85-TDP-35 Ab with full-length TDP-43. Conversely, Met 85 -TDP-35 immunoreactivity co-localized with TDP-43 pathology in motor neurons of ALS cases ( arrows ). Asterisk indicates lipofuscin. Scale bars 20 μm ( a – c ); 5 μm ( d )

Article Snippet: Equivalent amounts of protein samples were loaded onto 10 % SDS-PAGE gels and transferred to polyvinyldiflouride (PVDF) membranes, which were blocked in 5 % skim milk dissolved in Tris-buffered saline (TBS) containing 0.2 % Tween-20 for 1 h at RT, followed by overnight incubation at 4 °C with a polyclonal TDP-43 antibody (1:1000, Proteintech), a polyclonal N-terminal TDP-43 antibody (1:1000, Aviva Systems Biology), or a polyclonal C-terminal TDP-43 antibody (1:1000, generated in-house using a synthetic peptide, FGSSMDSKSSGWG, linked to keyhole limpet hemocyanin as the antigen).

Techniques: Immunohistochemical staining, Labeling, Immunofluorescence, Staining

Journal: Neural Regeneration Research

Article Title: Bone marrow mesenchymal stem cells repair spinal cord ischemia/reperfusion injury by promoting axonal growth and anti-autophagy

doi: 10.4103/1673-5374.141801

Figure Lengend Snippet: Effects of bone marrow mesenchymal stem cells on the expression of microtubule-associated light chain 3B (LC3B) and Beclin 1 in the spinal cord of rats with spinal cord ischemia/reperfusion injury. (A) Immunostaining for LC3B and Beclin 1 in the rat spinal cord (arrows show positive cells) (× 400). (B) Semi-quantitative analysis of LC3B and Beclin 1 immunostaining. n = 10 rats/group. Data were expressed as the mean ± SD. * P < 0.05, vs . control group; # P < 0.05, vs . sham surgery group; † P < 0.05, vs . model group (one-way analysis of variance followed by the Fisher's least significant difference test).

Article Snippet: Rabbit anti-growth associated protein-43 polyclonal antibody (1:1,000), rabbit anti-neurofilament-H (marker for mature neuronal axons) (Yabe et al., 2001) polyclonal antibody (1:1,000; Proteintech Group), rabbit anti-rat light chain 3B monoclonal antibody (1:1,000), or rabbit-Beclin 1 polyclonal antibody (1:1,000) were added overnight at 4°C.

Techniques: Expressing, Immunostaining

Journal: Neural Regeneration Research

Article Title: Bone marrow mesenchymal stem cells repair spinal cord ischemia/reperfusion injury by promoting axonal growth and anti-autophagy

doi: 10.4103/1673-5374.141801

Figure Lengend Snippet: Effect of bone marrow mesenchymal stem cells on the expression of growth associated protein-43 (GAP-43), neurofilament-H (NF-H), light chain 3B (LC3B), and Beclin 1 in the spinal cord of rats with spinal cord ischemia/reperfusion injury (western blot assay). Western immunoblots of GAP-43, NF-H, LC3B, and Beclin1 in the (I) control group, (II) sham surgery group, (III) model group, and (IV) stem cell therapy group. Data are expressed as the integrated optical density ratio of target protein to β-actin (mean ± SD). * P < 0.05, vs . con-trol group; # P < 0.05, vs . sham surgery group; † P < 0.05, vs . model group (one-way analysis of variance followed by the Fisher's least significant difference test).

Article Snippet: Rabbit anti-growth associated protein-43 polyclonal antibody (1:1,000), rabbit anti-neurofilament-H (marker for mature neuronal axons) (Yabe et al., 2001) polyclonal antibody (1:1,000; Proteintech Group), rabbit anti-rat light chain 3B monoclonal antibody (1:1,000), or rabbit-Beclin 1 polyclonal antibody (1:1,000) were added overnight at 4°C.

Techniques: Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Heat Shock-induced Phosphorylation of TAR DNA-binding Protein 43 (TDP-43) by MAPK/ERK Kinase Regulates TDP-43 Function *

doi: 10.1074/jbc.M116.753913

Figure Lengend Snippet: Antibody p-T153/Y155-TDP-43 specifically detects heat shock-mediated TDP-43 phosphorylation. A, immunoblots of HeLa cells treated with TDP-43-specific and control siRNA to compare levels of p-T153/Y155-TDP-43 following heat shock stress. Error bars, S.D. n = 4. B, detection of the heat shock-associated signal in SH-SY5Y cell lysate with p-T153/Y155-TDP-43 antibody blocked with a TDP-43 peptide corresponding to the Thr-153/Tyr-155 region (amino acids 148–161) phosphorylated at Thr-153 and Tyr-155 (T153P/Y155P), or with the corresponding non-phosphorylated peptide, as control. Two concentrations of peptides were used low (L) and high (H), described under “Experimental Procedures.” C, p-T153/Y155-TDP-43 detection of control and λ-phosphatase-treated lysates from control and heat shock-treated SH-SY5Y cells.

Article Snippet: The rabbit polyclonal antibodies recognizing p-T153/Y155 and p-Y155-TDP-43 were produced by 21st Century Biochemicals, MA.

Techniques: Phospho-proteomics, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Heat Shock-induced Phosphorylation of TAR DNA-binding Protein 43 (TDP-43) by MAPK/ERK Kinase Regulates TDP-43 Function *

doi: 10.1074/jbc.M116.753913

Figure Lengend Snippet: p-T153/Y155-TDP-43 detects nucleolar localization of TDP-43 in the dense coil-like structures. A, fluorescence imaging of non-treated HeLa cells showing total TDP-43 and p-T153/Y155-TDP-43 localization. B, p-T153/Y155 colocalization with the nucleolar marker fibrillarin in HeLa and SH-SY5Y cells as seen by confocal microscopy. C, detection of p-T153/Y155 in HeLa cells upon heat shock compared with control-treated cells. Bars, 10 μm.

Article Snippet: The rabbit polyclonal antibodies recognizing p-T153/Y155 and p-Y155-TDP-43 were produced by 21st Century Biochemicals, MA.

Techniques: Fluorescence, Imaging, Marker, Confocal Microscopy, Control

Journal: The Journal of Biological Chemistry

Article Title: Heat Shock-induced Phosphorylation of TAR DNA-binding Protein 43 (TDP-43) by MAPK/ERK Kinase Regulates TDP-43 Function *

doi: 10.1074/jbc.M116.753913

Figure Lengend Snippet: p-T153/Y155 is not associated with aggregation, stress granule recruitment, or clearance of TDP-43. A, fractionation of SH-SY5Y cells performed with control, heat shock-treated cells (43 °C for 30 min), and cells allowed to recover at 37 °C for 1 or 2 h (1 hr R, 2 hr R) following heat shock. Immunoblots of the total lysate (CL), RIPA (R), and urea (U) soluble fractions detecting p-T153/Y155 and total TDP-43. Equal volumes of total lysate and RIPA soluble fraction were loaded, whereas the urea fraction is 5-fold more concentrated. B, immunoblots of SH-SY5Y cells treated with the UPS inhibitor MG132 (20 μm for 5 h), heat shock (HS: 43 °C for 30 min), trehalose (100 mm for 24 h), thapsigargin (THP: 1 μm, 2 h), and serum starvation (24 h). C, fluorescence microscopy of control and heat shock-treated HeLa cells detecting p-T153/Y155 and a stress granule marker, TIAR. Arrows indicate TIAR-positive stress granules. D, control and MG132-treated HeLa cells (20 μm,5 h). Arrows indicate TDP-43 cytoplasmic aggregates detected with a phospho-independent antibody. Bars, 10 μm. E, control and trehalose (100 mm, 24 h) treated HeLa cells. Formation of autophagy vesicles (arrow, lower panels) visualized upon transfection with GFP-fused microtubule-associated protein 1A/1B-light chain 3 (LC3). Bars, 25 μm.

Article Snippet: The rabbit polyclonal antibodies recognizing p-T153/Y155 and p-Y155-TDP-43 were produced by 21st Century Biochemicals, MA.

Techniques: Fractionation, Control, Western Blot, Fluorescence, Microscopy, Marker, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Heat Shock-induced Phosphorylation of TAR DNA-binding Protein 43 (TDP-43) by MAPK/ERK Kinase Regulates TDP-43 Function *

doi: 10.1074/jbc.M116.753913

Figure Lengend Snippet: TDP-43 is specifically phosphorylated by MEK at Thr-153/Tyr-155. A, human TDP-43 amino acid sequence surrounding Thr-153 and Tyr-155 (underlined) aligned with the human ERK1 activation loop sequence where MEK phosphorylates at Thr-202 and Tyr-204 (underlined). B, immunoblots of heat shock and control treated HEK-293 and SH-SY5Y cells. The levels of phospho-independent proteins are shown and tubulin was used as loading control. C, SH-SY5Y cells treated with specific MEK inhibitors PD184352 (10 μm) and PD98059 (50 μm), and with a specific inhibitor of the downstream kinase ERK FR180204 (30 μm). Cells were exposed to heat shock following 1 h of inhibitor treatment. D, SH-SY5Y cells treated with the protein phosphatase PP1/2A inhibitor, okadaic acid (0.5 μm), combined with the MEK inhibitor PD184352 for 1 h before heat shock. E, p-T153/Y155 levels analyzed upon expression of the constitutively active GFP-MEK_DD mutant and a GFP control construct in SH-SY5Y cells in the absence of heat shock.

Article Snippet: The rabbit polyclonal antibodies recognizing p-T153/Y155 and p-Y155-TDP-43 were produced by 21st Century Biochemicals, MA.

Techniques: Sequencing, Activation Assay, Western Blot, Control, Expressing, Mutagenesis, Construct