ddk rabbit monoclonal antibody, recognizing both n- and c-terminal tags  (OriGene)

Journal: Frontiers in Pharmacology

Article Title: A peptide derived from TID1S rescues frataxin deficiency and mitochondrial defects in FRDA cellular models

doi: 10.3389/fphar.2024.1352311

Figure Lengend Snippet: TID1 physically interacts with frataxin both in vivo in mouse cortex and in vitro in cortical neurons. In both cortical homogenates (A) and neuronal lysates (B) , frataxin was immunoprecipitated by a TID1L/S antibody but not control IgG. Similarly, a specific TID1L antibody but not control IgG immunoprecipitated frataxin from cortical homogenates (C) and HEK293 cells (D) but to a much lesser extent. In an in vitro binding assay, purified glutathione S- transferase (GST)-frataxin but not purified GST pulled down TID1L (E) . In HEK293 cells transfected with frataxin-HA and TID1S-Flag, immunofluorescence was performed using anti-HA and anti-Flag antibodies. Frataxin colocalized with TID1S (F) . Frataxin also colocalized with endogenously expressed TID1L stained with an anti-TID1L antibody (F) , further supported the interaction between TID1 and frataxin.

Article Snippet: After 4 washes, 2 μg human TID1L fused to a C-terminal Myc/DDK tag (Origene, Rockville, MD, Catalog #TP315039) was added and incubated overnight at 4°C.

Techniques: In Vivo, In Vitro, Immunoprecipitation, Control, Binding Assay, Purification, Transfection, Immunofluorescence, Staining

Journal: Frontiers in Pharmacology

Article Title: A peptide derived from TID1S rescues frataxin deficiency and mitochondrial defects in FRDA cellular models

doi: 10.3389/fphar.2024.1352311

Figure Lengend Snippet: TID1L protein levels are reduced in FRDA patient-derived cells. FRDA patient buccal cells, skin fibroblasts, platelets or PBMCs were lysed and subjected to Western blotting with the indicated antibodies. The amount of immunoreactivity in the lysates was quantified as a percentage of the controls. Representative blots and bar graphs demonstrate reduced frataxin and TID1L in buccal cells (A,B) ( n = 14 for controls and n = 16 for patients), platelets (C,D) ( n = 16 for controls and n = 17 for patients) and fibroblasts (G,H) ( n = 15 for controls and n = 20 for patients). While frataxin was significantly reduced in PBMCs, no change in TID1L was detected in PBMCs (E,F) ( n = 18 for both controls and patients). ∗ p < 0.05; ∗∗ p < 0.01. Data were shown as mean ± SE.

Article Snippet: After 4 washes, 2 μg human TID1L fused to a C-terminal Myc/DDK tag (Origene, Rockville, MD, Catalog #TP315039) was added and incubated overnight at 4°C.

Techniques: Derivative Assay, Western Blot

Journal: Frontiers in Pharmacology

Article Title: A peptide derived from TID1S rescues frataxin deficiency and mitochondrial defects in FRDA cellular models

doi: 10.3389/fphar.2024.1352311

Figure Lengend Snippet: Effect of TID1 overexpression on frataxin protein levels. HEK293 cells transfected with frataxin and TID1 plasmid DNAs were lysed and subjected to Western blotting with the indicated antibodies. The amount of immunoreactivity in the lysates was quantified as a percentage of vector control. Representative blots and bar graphs demonstrate increased frataxin precursor and decreased intermediate and mature frataxin following TID1L or TID1S overexpression (A,B) ( n = 8). TID1L H121Q and TID1S H121Q had similar effects as wildtype TID1L and TID1S (A,B) ( n = 6), respectively. No change was found in GRP75 levels upon TID1L or TID1S overexpression (C) . ∗ p < 0.05, ∗∗ p < 0.01. Data were shown as mean ± SE.

Article Snippet: After 4 washes, 2 μg human TID1L fused to a C-terminal Myc/DDK tag (Origene, Rockville, MD, Catalog #TP315039) was added and incubated overnight at 4°C.

Techniques: Over Expression, Transfection, Plasmid Preparation, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: A peptide derived from TID1S rescues frataxin deficiency and mitochondrial defects in FRDA cellular models

doi: 10.3389/fphar.2024.1352311

Figure Lengend Snippet: Mechanistic study of TID1 overexpression-caused changes in frataxin levels. HEK293 cells transfected with frataxin and TID1 plasmid DNAs were subject to mitochondria fractionation followed by Western blotting analysis. Frataxin precursor is predominantly localized in the mitochondrial fraction upon TID1L or TID1S overexpression (A) . Tom20 was used as a mitochondrial marker (A) . Treatment with MG132 (10 μM) also had no effect on TID1L or TID1S overexpression-caused decrease in intermediate and mature frataxin (B) . Frataxin G130V mutant transfected HEK293 cells were used as a positive control (B) . Neither TID1L nor TID1S interacted with MPP, in contrast to GRP75 (C) .

Article Snippet: After 4 washes, 2 μg human TID1L fused to a C-terminal Myc/DDK tag (Origene, Rockville, MD, Catalog #TP315039) was added and incubated overnight at 4°C.

Techniques: Over Expression, Transfection, Plasmid Preparation, Fractionation, Western Blot, Marker, Mutagenesis, Positive Control

Journal: Frontiers in Pharmacology

Article Title: A peptide derived from TID1S rescues frataxin deficiency and mitochondrial defects in FRDA cellular models

doi: 10.3389/fphar.2024.1352311

Figure Lengend Snippet: TID1S overexpression decreases mature frataxin in human skin fibroblasts. Human skin fibroblasts from healthy individuals were transduced with lentivirus carrying pHAGE-TID1S gene or vector control for 5 days before Western blotting or immunofluorescence. TID1S transduction decreased mature frataxin (A, B) and ATP levels (C) ( n = 5). TID1S transduction also caused mitochondria fractionation in fibroblasts (D) . Vector control was stained with an anti-GFP antibody and TID1S was stained with an anti-TID1L/S antibody. ∗ p < 0.05, ∗∗ p < 0.01. Data were shown as mean ± SE.

Article Snippet: After 4 washes, 2 μg human TID1L fused to a C-terminal Myc/DDK tag (Origene, Rockville, MD, Catalog #TP315039) was added and incubated overnight at 4°C.

Techniques: Over Expression, Transduction, Plasmid Preparation, Control, Western Blot, Immunofluorescence, Fractionation, Staining

Journal: eLife

Article Title: Tmem263 deletion disrupts the GH/IGF-1 axis and causes dwarfism and impairs skeletal acquisition

doi: 10.7554/eLife.90949

Figure Lengend Snippet: ( A ) Sequence alignment of full-length human (NP_689474), mouse (NP_001013046), chicken (NP_001006244), Xenopus frog (NP_989399), and zebrafish (NP_998306) transmembrane protein 263 (TMEM263) using Clustal-omega . Identical amino acids are shaded black and similar amino acids are shaded gray. Gaps are indicated by dash lines. The two predicted transmembrane domains are indicated in red. ( B ) TMEM263 expression across normal human tissues based on the consensus Human Protein Atlas (HPA) and Gene-Tissue Expression (GTEx) datasets. The data can be accessed via the HPA database ( https://www.proteinatlas.org ). nTPM denotes normalized protein-coding transcripts per million and it corresponds to the mean values of the different individual samples from each tissue. Bars are color-coded based on tissue groups with functional features in common. ( C ) Tmem263 expression across mouse tissues (n=11). Relative expression across tissues were first normalized to β-actin , then normalize to the tissue (pancreas) with the lowest expression. ( D ) Immunoblot analysis of cell lysate from HEK293 cells transfected with a control pCDNA3 empty plasmid or plasmid encoding human TMEM263 tagged with a C-terminal Myc-DDK epitope. Immunoblots were probed with an anti-FLAG (DDK) antibody (left panel) or an anti-TMEM263 antibody (right panel). ( E ) TMEM263 is localized to the plasma membrane. Surface biotinylation was carried out on transfected HEK293 cells. Biotinylated plasma membrane proteins were captured with Avidin-agarose beads, eluted, and immunoblotted for TMEM263 with an anti-FLAG antibody. Figure 1—source data 1. Top left – Original uncropped membrane from imager showing blue channel only as a black and white image. Two close molecular markers are noted. FLAG-tagged TMEM263 at the predicted molecular weight is marked. The cropped region used for the main figure image is marked with a dotted box. This membrane was probed with the anti-FLAG antibody. Bottom left – The same membrane as shown above, but overexposed and imaged with the light channel to show the entire membrane outline. The anti-FLAG antibody is very clean and therefore the membrane boarder is hard to see otherwise. Top right – Original uncropped membrane from imager showing blue channel only as a black and white image. Two close molecular markers are noted. FLAG-tagged TMEM263 at the predicted molecular weight is marked. The cropped region used for the main figure image is marked with a dotted box. This membrane was probed with the anti-FLAG antibody. Bottom left – The same membrane as shown above, but overexposed and imaged with the light channel to show the entire membrane outline. The anti-TMEM263 antibody is fairly clean and therefore the membrane boarder is hard to see otherwise. Figure 1—source data 2. Left – Original uncropped membrane from imager showing blue channel only as a black and white image. Two close molecular markers are noted. FLAG-tagged TMEM263 at the predicted molecular weight is marked. The cropped region used for the main figure image is marked with a dotted box. This membrane was probed with the anti-FLAG antibody. Right – The same membrane as on the left, but overexposed and imaged with the light channel to show the entire membrane outline. The anti-FLAG antibody is very clean and therefore the membrane boarder is hard to see otherwise.

Article Snippet: Mammalian expression plasmid encoding human TMEM263 with a C-terminal epitope tag (Myc-DDK) was obtained from Origene (RC203933).

Techniques: Sequencing, Expressing, Functional Assay, Western Blot, Transfection, Control, Plasmid Preparation, Membrane, Avidin-Biotin Assay, Molecular Weight

Journal: eLife

Article Title: Tmem263 deletion disrupts the GH/IGF-1 axis and causes dwarfism and impairs skeletal acquisition

doi: 10.7554/eLife.90949

Figure Lengend Snippet:

Article Snippet: Mammalian expression plasmid encoding human TMEM263 with a C-terminal epitope tag (Myc-DDK) was obtained from Origene (RC203933).

Techniques: Knock-Out, Generated, Transgenic Assay, Transfection, Construct, Expressing, Plasmid Preparation, FLAG-tag, Recombinant, Injection, Isolation, Enzyme-linked Immunosorbent Assay, SYBR Green Assay

Journal: bioRxiv

Article Title: Tmem263 deletion disrupts the GH/IGF-1 axis and causes dwarfism and impairs skeletal acquisition

doi: 10.1101/2023.08.02.551694

Figure Lengend Snippet: (A) Sequence alignment of full-length human (NP_689474), mouse (NP_001013046), chicken (NP_001006244), xenopus frog (NP_989399), and zebrafish (NP_998306) transmembrane protein 263 (TMEM263) using Clustal-omega . Identical amino acids are shaded black and similar amino acids are shaded grey. Gaps are indicated by dash lines. The two predicted transmembrane domains are indicated in red. (B) TMEM263 expression across normal human tissues based on the consensus Human Protein Atlas (HPA) and Gene-Tissue Expression (GTeX) datasets. The data can be accessed via the HPA database ( www.proteinatlas.org ). nTPM denotes normalized protein-coding transcripts per million and it corresponds to the mean values of the different individual samples from each tissue. Bars are color-coded based on tissue groups with functional features in common. (C) Tmem263 expression across mouse tissues ( n = 11 ). Relative expression across tissues were first normalized to β-actin , then normalize to the tissue (pancreas) with the lowest expression. (C) Immunoblot analysis of cell lysate from HEK293 cells transfected with a control pCDNA3 empty plasmid or plasmid encoding human TMEM263 tagged with a C-terminal Myc-DDK epitope. Immunoblots were probed with an anti-FLAG (DDK) antibody (left panel) or an anti-TMEM263 antibody (right panel). (D) TMEM263 is localized to the plasma membrane. Surface biotinylation was carried out on transfected HEK293 cells. Biotinylated plasma membrane proteins were captured with Avidin-agarose beads, eluted, and immunoblotted for TMEM263 with an anti-FLAG antibody.

Article Snippet: Mammalian expression plasmid encoding human TMEM263 with a C-terminal epitope tag (Myc-DDK) was obtained from Origene (RC203933).

Techniques: Sequencing, Expressing, Functional Assay, Western Blot, Transfection, Control, Plasmid Preparation, Clinical Proteomics, Membrane, Avidin-Biotin Assay

Journal: bioRxiv

Article Title: Tmem263 deletion disrupts the GH/IGF-1 axis and causes dwarfism and impairs skeletal acquisition

doi: 10.1101/2023.08.02.551694

Figure Lengend Snippet: (A) Generation of Tmem263 knockout (KO) mice. The exon 3 that encodes ~81% of the full-length protein was deleted using CRISPR/Cas9 method and confirmed with DNA sequencing. The location and sequence of the two guide RNAs (gRNA) used to generate the deletion were underlined. Filled-in black boxes indicate part of the exon that codes for Tmem263 protein, and white boxes indicate part of the exon that codes for 5’ and 3’ UTR of the transcript. (B) Wild-type (WT) and KO alleles were confirmed by PCR genotyping. (C) The complete loss of Tmem263 transcript in KO mice was confirmed by qPCR in male and female mouse liver and hypothalamus (WT, n = 6-8; KO, n = 6-8). (D) The expected Mendelian versus observed genotype distributions in P1 pups ( n = 82). (E) Representative images of WT and Tmem263 -KO pups at postnatal day 1 (P1). Milk spots are indicated by a red arrow. (F) Representative Alcian blue and Alizarin red staining of axial skeletal and cartilage in WT and KO P1 pups. (G) Body weights of WT and Tmem263 -KO pups at P1 (WT = 17; Het = 42; KO = 23), P7 (WT = 15; het = 28; KO = 5), P14 (WT = 16; het = 34; KO = 4), and P21 (WT = 33; het = 41; KO = 13). For panel G, we combined the data of male and female pups from P1 to P21. (H) Representative images of adult WT and Tmem263 -KO mice at 9 weeks of age. (I-J) Body weights and body length of WT (+/+), heterozygous (+/−), and KO (−/−) male and female mice at 9 weeks of age. Sample size for males (WT = 45; het = 73; KO = 9) and females (WT = 30; het = 59; KO = 8). (K) The growth curve trajectory based on the combined data in G and I. All data are presented as mean ± S.E.M. **** P < 0.0001 (One-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: Mammalian expression plasmid encoding human TMEM263 with a C-terminal epitope tag (Myc-DDK) was obtained from Origene (RC203933).

Techniques: Knock-Out, CRISPR, DNA Sequencing, Sequencing, Staining

Journal: bioRxiv

Article Title: Tmem263 deletion disrupts the GH/IGF-1 axis and causes dwarfism and impairs skeletal acquisition

doi: 10.1101/2023.08.02.551694

Figure Lengend Snippet: (A) Representative microCT images of bone (femur) showing a dramatic reduction in size in Tmem263 KO (−/−) mice relative to WT (+/+) and heterozygous (+/−) controls at 8 weeks of age. (B) Femur length of WT (+/+), heterozygous (+/−), and KO (−/−) male and female mice at 8 weeks of age. (C) Quantification of trabecular bone volume per tissue volume (BV/TV) in the distal femur of WT (+/+), heterozygous (+/−), and KO (−/−) male and female mice. (D) Quantification of trabecular number (Tb. N) in the distal femur. (E) Trabecular bone thickness (Tb. Th). (F) Cortical tissue area (Tt. Ar). (G) Cortical area per tissue area. (H) Cortical thickness. (I) Tissue area per femur length. (J) Cortical thickness per femur length in male and female mice. (K) Representative images of tibial growth plate histology in WT and Tmem263 -KO male mice. (L-N) Quantification of growth plate length (L), proliferative zone length (M), and hypertrophic zone length (N) in WT ( n = 10) and KO ( n = 10) male mice. All data were collected on 8-week-old mice. Sample size for panel B-J: males (WT = 6; het = 6; KO = 11) and females (WT = 6; het = 6; KO = 9). All data are mean ± S.E. ** P < 0.01; *** P < 0.001; **** P < 0.0001 (One-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: Mammalian expression plasmid encoding human TMEM263 with a C-terminal epitope tag (Myc-DDK) was obtained from Origene (RC203933).

Techniques:

Journal: bioRxiv

Article Title: Tmem263 deletion disrupts the GH/IGF-1 axis and causes dwarfism and impairs skeletal acquisition

doi: 10.1101/2023.08.02.551694

Figure Lengend Snippet: Serum levels of growth hormone (GH; A), IGF-1 (B), IGFBP3 (C), IGFALS (D), insulin (E), glucose (F), calcium (G), and phosphate (H) in WT (+/+), heterozygous (+/−) and Tmem263 -KO (−/−) male and female mice at 8 weeks old. Sample size for panel A (GH): males (WT = 9; het = 9; KO = 5) and females (WT = 10; het = 7; KO = 8). Panel B (IGF-1): males (WT = 9; het = 9; KO = 9) and females (WT = 10; het = 7; KO = 7). Panel C (IGFBP3): males (WT = 15; het = 16; KO = 11) and females (WT = 12; het = 18; KO = 10). Panel D (IGFALS): males (WT = 10; het = 10; KO = 8) and females (WT = 10; het = 10; KO = 8). Panel E (insulin): males (WT = 12; het = 17; KO = 8) and females (WT = 10; het = 16; KO = 8). Panel F (glucose): males (WT = 9; het = 14; KO = 9) and females (WT = 6; het = 15; KO = 8). Panel G and H (calcium and phosphate): males (WT = 10; het = 10; KO = 8) and females (WT = 10; het = 10; KO = 8). (I) Ratio of calcium-to-phosphate in WT, heterozygous, and KO male and female mice. Sample size for males (WT = 10; het = 10; KO = 8) and females (WT = 10; het = 10; KO = 8). All data are presented as mean ± S.E.M. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 (One-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: Mammalian expression plasmid encoding human TMEM263 with a C-terminal epitope tag (Myc-DDK) was obtained from Origene (RC203933).

Techniques:

Journal: bioRxiv

Article Title: Tmem263 deletion disrupts the GH/IGF-1 axis and causes dwarfism and impairs skeletal acquisition

doi: 10.1101/2023.08.02.551694

Figure Lengend Snippet: (A) Volcano plot of male mouse liver transcriptome. (B) Enrichment analysis of differentially expressed genes (DEGs) by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome ( http://www.reactome.org ) databases. Plots of -Log(adj. p) vs gene ratio to show complete spread of all enrichment results for each analysis. The most effected categories are highlighted and labeled with the category name and number of up- and down-regulated genes within each. (C) Volcano plot showing the expression of all transcription factor (TF) genes detected in the male mouse liver transcriptome. TFs that are significantly up- or down-regulated in KO male mouse liver are highlighted. (D) Heat map of DEGs involved in growth and metabolism. (E) Heat map of all protein-coding DEGs from the cytochrome P450 (Cyp) gene family. (F) Heat map of all protein-coding DEGs from the Major urinary protein (Mup) gene family. (G-I) Tmem263 -KO male liver DEG overlap comparison to three separate public data sets of mouse liver gene expression: (G) WT male vs. WT female mice , (H) Hypophysectomized vs. sham control male mice , and (I) Stat5b -KO vs WT male mice . (J) Summary of key findings underpinning the dwarfism and skeletal dysplasia phenotypes of Tmem263-null mice. All heat map data is shown on a column z-score scale. Only significantly different genes (adjusted p-value < 0.05 and Log 2 (FC) <−1 or >1) are shown for all heat maps. PCG = protein coding gene, NPCG = non-protein coding gene. Sample size for male WT ( n = 8) and KO (n = 8) mice were 9 weeks old for all data shown.

Article Snippet: Mammalian expression plasmid encoding human TMEM263 with a C-terminal epitope tag (Myc-DDK) was obtained from Origene (RC203933).

Techniques: Labeling, Expressing, Comparison, Gene Expression, Control