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Antibodies

ATG13 phospho S318 Antibody

Product Sizes
100 ug
600-401-C49-100UG
About this Product
SKU:
600-401-C49
Additional Names:
ATG13|rabbit anti-ATG13 pS318 Antibody, ATG-13, ATG 13, Autophagy-related protein 13, KIAA0652
Application:
Detection/Quantitation, Dot Blot, ELISA, Flow Cytometry, Immunofluorescence, Western Blot
Buffer:
0.02 M Potassium Phosphate, 0.15 M Sodium Chloride
CE/IVD:
RUO
Clonality:
Polyclonal
Extra Details:
ATG13 is a target of the TOR kinase signaling pathway that regulates autophagy through the control of the phosphorylation status of ATG13 and ULK1 through their stable complex, and the regulation of ATG13-ULK1-RB1CC1. ATG13 also forms a stable complex with FIP200. Ulk1 phosphorylates ATG13 on S318 and promotes its release to damaged mitochondria. Autophagy is a normal process in eukaryotes required for turnover of cellular components during starvation and stress. It plays an essential role in cellular differentiation, cell death and aging. Defects in this evolutionarily conserved process may contribute to certain human diseases such as cancer, neurodegenerative diseases, muscular disorders and pathogen infections. ATG13 is one of several ATG genes required for autophagosome formation in mammalian cells. mTOR interacts with this complex in a nutrient dependent manner and phosphorylates Atg13 and ULK1.
Formulation:
0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2
Host:
Rabbit
Immunogen:
Anti-ATG13 pS318 antibody was prepared by repeated immunizations with a synthetic peptide corresponding to the region near S318 of ATG13.
Isotype:
IgG
Physical State:
Liquid
Reactivities:
Human
Shipping Conditions:
Dry Ice
Specificity:
This affinity-purified antibody is directed against the phosphorylated form of human ATG13 protein at the pS318 residue. The product was affinity purified from monospecific antiserum by immunoaffinity purification. Antiserum was first purified against the phosphorylated form of the immunizing peptide. The resultant affinity purified antibody was then cross adsorbed against the non-phosphorylated f
Storage Conditions:
-20[o]C aliquoted. Aliquot. Avoid freeze/thaw cycles.
Supplier:
Rockland Inc
Type:
Antibody: Polyclonal Antibody
Western blot using Rockland's affinity purified anti-ATG13 pS318 antibody shows detection of phosphorylated ATG13 in 293T cells engineered to coexpress Ulk1 and Atg13 (Ulk1 + Atg13). In the left lane was loaded kinase-dead hypophosphorylated Ulk1-K46A mutant + ATG13. The right lane contains the 293T Ulk1 + ATG13 lysate and shows detection at approximately 57 kDa. The antibody was purified and resolved by SDS-PAGE, then transferred to nitrocellulose membrane. The membrane was blocked with 5% Blotto (p/n B501-0500) and probed with the primary antibody at 1ug/mL overnight at 4°C. After washing, the membrane was probed with Goat Anti-Rabbit HRP secondary 1:5000 in detection buffer (p/n MB-070) for 45 minutes at room temperature. In collaboration with Charles Dorsey at Eli Lilly, Indianapolis, IN and John Cleveland at Scripps, Jupiter, FL.
Western Blot of Rabbit Anti-ATG13pS318 Antibody. Lane 1: Opal Prestained Molecular Weight Marker (p/n MB-210-0500). Lane 2: BSA Conjugated ATG13 pS318 peptide - reduced [0.2ug]. Lane 3: BSA Conjugated ATG13 S318 peptide - reduced [0.2ug]. Primary Antibody: Anti-ATG13pS318 [Rabbit] Antibody at 1.0ug/mL overnight at 2-8°C. Secondary Antibody: Anti-Rabbit IgG [Goat] Peroxidase conjugated (p/n 611-1302) at 1:40,000 for 30mins at RT. Block: Blocking Buffer for Fluorescent Western Blotting (p/n MB-070) for 1hr at RT. Expected: detects the P-peptide and does not detect the NP-peptide. Exposure: 6.1sec.
Autophagic vesicles are locally formed in dendrites of cultured neurons following LTD. A) Top, representative super-resolution microscopy dSTORM image of a secondary dendrite labeled with an antibody against LC3, 15 min after cLTD. Bottom, magnification of representative U-shaped LC3-positive structures in dendrites, 15 min after NMDA or DHPG pulses. Scale bars: 2 um and 250 nm, as indicated. (N = 3 independent experiments). B) Graph showing the number of LC3-positive U-shaped structures in secondary dendrites visualized in (a), before (control) and 15 min or 60 min after NMDAR- and mGluR-LTD. Bars represent mean values +/- SEM. N = 3 independent experiments per condition (n > 9 dendrites per condition). Statistical analysis was performed by one-way ANOVA. For the time point of 15 min F(2, 33) = 17.93, p < 0.0001) (Tukey's test Pcontrol-NMDA < 0.0001, Pcontrol-DHPG < 0.0001). For the time point of 60 min F(2, 21) = 9.459, p = 0.0012) (Tukey's test Pcontrol-NMDA = 0.0041, Pcontrol-DHPG = 0.0024). C) Graph showing the size distribution (nm) of the dendritic U-shaped LC3-positive structures visualized in a upon NMDAR- and mGluR-LTD. Bars represent mean values +/- SEM for each analysed dendrite. N = 3 independent experiments (n > 40 dendrites per condition). D) Confocal images of dendrites immunolabeled with antibodies against WIPI2, LC3, and MAP2 before (control) or after 15 min of NMDAR- and mGluR-LTD. Scale bar: 10 um. (N = 6 independent experiments). E) Representative confocal images of neurons immunolabeled with antibodies against ULK1, Atg101, Atg13, FIP200 and, along with MAP2 to label dendrites before (control) or 15 min after LTD-inducing pulses. Scale bar: 20 um. Graphs showing the number of puncta positive for each ULK1-complex component in secondary dendrites, normalized for dendrite length, in every condition, as indicated. Graph bars represent mean values +/- SEM. N = 6 independent experiments per condition. Statistical analyses were performed using one-way ANOVA. ULK1: F(2,15) = 24.48, P < 0.0001 (Tukey's multiple comparison test, Pcontrol_NMDAR < 0.0001, Pcontrol_mGluR < 0.0001, PNMDAR_mGluR = 0.8825). Atg101: F(2,15) = 24.31, P < 0.0001 (Tukey's multiple comparison test, Pcontrol_NMDAR < 0.0001, Pcontrol_mGluR < 0.0001, PNMDAR_mGluR = 0.9329). Atg13: F(2,15) = 8.386, P = 0.0036 (Tukey's multiple comparison test, Pcontrol_NMDAR = 0.007, Pcontrol_mGluR = 0.0086, PNMDAR_mGluR = 0.9940). FIP200: F(2,15) = 17.66, P = 0.0001 (Tukey's multiple comparison test, Pcontrol_NMDAR = 0.0002, Pcontrol_mGluR = 0.0009, PNMDAR_mGluR = 0.6440). F) Western blot analyses for Atg13, FIP200, ULK1, Atg101, LC3, and B Beta-III tubulin (Tuj1) in neuronal lysates, under control conditions and 15 min after NMDAR- and mGluR-LTD. Graphs showing the normalized protein levels of Atg13, FIP200, ULK1, Atg101 under the aforementioned conditions. Bars represent mean values +/- SEM. N = 3 independent experiments for ULK1 complex proteins, N = 5 independent experiments for LC3-II. Statistical analyses were performed using one-way ANOVA. ULK1: F(2,6) = 13.57, P = 0.0059 (Tukey's multiple comparison test Pcontrol_NMDA = 0.0056, Pcontrol_DHPG = 0.0253). Atg101: F(2,6) = 27.57, P = 0.0009 (Tukey's multiple comparison test Pcontrol_NMDA = 0.0013, Pcontrol_DHPG = 0.79). Atg13: F(2,6) = 113.1, P < 0.0001 (Tukey's multiple comparison test Pcontrol_NMDA = 0.0027, Pcontrol_DHPG < 0.0001). FIP200: F(2,6) = 15.14, P = 0.0045 (Tukey's multiple comparison test Pcontrol_NMDA = 0.0240, Pcontrol_DHPG = 0.041). LC3-II: F (2, 12) = 4,969, P = 0.0268 (Tukey's multiple comparison test Pcontrol_NMDA = 0.0478, Pcontrol_DHPG = 0.0421). Figure provided by CiteAb. Source: Nat Commun, PMID: 35115539.
Restored regulation of ULK1 protein levels in SMCR8 knockout cells.(A,B) Early (A) or late (B) passages of HAP1 SMCR8 wildtype (wt) or knockout (ko) cells were subjected to SDS-PAGE and immunoblot with indicated antibodies. PCNA served as loading control. (C) 293T SMCR8 wildtype (wt) or knockout (ko) cells were analyzed as in (A). (D) 293 T cells were transfected with non-targeting control (sicon) or SMCR8 siRNA. Half of the cells were re-transfected every 2-3 days with non-targeting control (sicon) or SMCR8 siRNA while the other half was harvested and subjected to SDS-PAGE and immunoblotting with indicated antibodies. PCNA served as loading control.DOI:http://dx.doi.org/10.7554/eLife.23063.027 Figure provided by CiteAb. Source: Elife, PMID: 28195531.
SMCR8 regulates ULK1 gene expression.(A) Lysates from 293 T cells transfected with non-targeting control (sicon) or SMCR8 siRNA were subjected to SDS-PAGE and immunoblotting with indicated antibodies. PCNA served as loading control. (B) Lysates from 293 T cells transfected with non-targeting control (sicon) or SMCR8 siRNA as well as with HA-tagged SMCR8 were subjected to SDS-PAGE and immunoblotting with indicated antibodies. PCNA served as loading control. exp. = exposure. (C) Lysates from 293 T cells transfected with indicated siRNAs and grown in absence (DMSO) or presence of 250 nM Torin1 for 2 hr were lysed and analyzed as in (A). Vinculin served as loading control. (D,E) 293T (D) or U2OS (E) cells were transfected with indicated siRNAs for 72 hr prior to RNA isolation, preparation of cDNA and RT-qPCR with ULK1, FIP200 and SMCR8 specific primers. Error bars represent SEM. Significance was determined using two-way ANOVA compared with sicon. All experiments were performed n = 3.DOI:http://dx.doi.org/10.7554/eLife.23063.026Restored regulation of ULK1 protein levels in SMCR8 knockout cells.(A,B) Early (A) or late (B) passages of HAP1 SMCR8 wildtype (wt) or knockout (ko) cells were subjected to SDS-PAGE and immunoblot with indicated antibodies. PCNA served as loading control. (C) 293T SMCR8 wildtype (wt) or knockout (ko) cells were analyzed as in (A). (D) 293 T cells were transfected with non-targeting control (sicon) or SMCR8 siRNA. Half of the cells were re-transfected every 2-3 days with non-targeting control (sicon) or SMCR8 siRNA while the other half was harvested and subjected to SDS-PAGE and immunoblotting with indicated antibodies. PCNA served as loading control.DOI:http://dx.doi.org/10.7554/eLife.23063.027 Figure provided by CiteAb. Source: Elife, PMID: 28195531.