Rabbit anti atg5 antibody

Manufactured by Cell Signaling Technology

Sourced in United States

Rabbit anti-ATG5 antibody is a primary antibody that recognizes the ATG5 protein, which plays a crucial role in the autophagy process. This antibody can be used for the detection and analysis of ATG5 expression in various research applications.

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5 protocols using rabbit anti atg5 antibody

Autophagy and Cell Cycle Regulation Assay

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The primary antibodies used in this study included the rabbit anti-Atg5 antibody (Cell signaling, cat:12994), rabbit anti-LC-3 antibody (MBL, cat: PM036), rabbit anti-p62 antibody (Cell Signaling, cat:5114), rabbit anti-actin antibody (Abcam, cat: ab8227), rabbit anti-Beclin-1 antibody (Abcam. ab51031), rabbit anti-Cyclin-D1 (Cell Signaling, cat:2978), rabbit anti-Cyclin-A2 (Cell Signaling, cat:4656), rabbit anti-Cyclin-E1 (Cell Signaling, cat:4129), rabbit anti-Cyclin-B1 (Cell Signaling, cat:4138), rabbit anti-p53 (Cell Signaling, cat:2524), rabbit anti-p21 (Abcam, ab7960), rabbit anti-p27 (Abcam, ab7961), rabbit anti-Caspase 8 (Cell Signaling, cat:9496), rabbit anti-Caspase 9 (Cell Signaling, cat:9509), rabbit anti-Caspase 3 (Cell Signaling, cat:9662), rabbit anti-Cytochrom C (Cell Signaling, cat:4272), rabbit anti-COX IV (Cell Signaling, cat:4844).

Chen Y., Xu Z., Zeng Y., Liu J., Wang X, & Kang Y. (2021). Altered metabolism by autophagy defection affect liver regeneration. PLoS ONE, 16(4), e0250578.

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Immunoblotting of Autophagy Proteins

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The primary antibodies used in this study included the mouse anti-STX7 antibody (Sigma-Aldrich), rabbit anti-ATG5 antibody (Cell Signaling), rabbit anti-p62 antibody (Cell Signaling), mouse anti-HCV NS5A monoclonal antibody (Millipore), rabbit anti-LC3 antibody (Sigma-Aldrich), and rabbit anti-calnexin antibody (Abcam). Proteins were extracted from cell lysates for western-blot analysis using the M-PER mammalian protein extraction reagent (Thermo Fisher Scientific) following the manufacturer’s protocol. The ER Tracker Blue was purchased from Thermo Fisher Scientific.

Wang L., Kim J.Y., Liu H.M., Lai M.M, & Ou J.H. (2017). HCV-induced autophagosomes are generated via homotypic fusion of phagophores that mediate HCV RNA replication. PLoS Pathogens, 13(9), e1006609.

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Confirming ATG5 Knockdown by Western Blot

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Western blot analysis was performed to confirm ATG5 knockdown. Primary GECs at 80% confluence were transfected in serum free KGM medium (Lonza) with 5 pmol of pre-designed ATG5 siRNA duplexes (siRNA ID#: s18160; Ambion) using lipofectamine RNAiMAX Reagent (Invitrogen) for 48 hours or Non-target pool siRNA (Dharmacon). Colorimetric Bradford Assay (Bio-Rad) was used to determine protein concentrations of the transfected and samples. Equal amount of protein samples were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After gel electrophoresis, the proteins were transferred onto a nitrocellulose membrane using wet-transfer system and the membrane was blocked in Tris-buffered saline with 0.1% Tween 20 (TBST) containing 5% nonfat dry milk. The membrane was then incubated with rabbit anti-ATG5 antibody at a dilution of 1:500 (Cell Signaling) and treated with anti-rabbit HRP-conjugated secondary antibody at 1:1000 (Cell Signaling). The blot was then stripped and probed with mouse anti-ß-tubulin antibody 1:1000 and anti-mouse HRP-conjugated secondary antibody (Cell Signaling) at 1:1000. Protein bands were visualized using enhanced chemiluminescence (ECL, GE Healthcare) and band intensities were examined using NIH ImageJ.

Lee K., Roberts J.S., Choi C.H., Atanasova K.R, & Yilmaz Ö. (2018). Porphyromonas gingivalis traffics into endoplasmic reticulum-rich-autophagosomes for successful survival in human gingival epithelial cells. Virulence, 9(1), 845-859.

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Autophagy Regulation Mechanisms Elucidation

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Bafilomycin A1 (Baf A1, 20 μM) was purchased from Selleck (USA), and the classical autophagy inhibitor 3-methyladenine (3-MA, 1 mM) was purchased from Sigma-Aldrich (USA). Protein A/G-Sepharose (Santa Cruz, USA) was used in the immunoprecipitation assays. The antibodies used in this study included rabbit anti-MPR (cation independent) antibody (Abcam, USA), rabbit anti-α-Tubulin antibody (Proteintech, USA), rabbit anti-cathepsin B antibody (Santa Cruz, USA), mouse anti-cathepsin D antibody (Santa Cruz, USA), rabbit anti-Lamp1 antibody (Abcam, USA), rat anti-Lamp1 antibody (Abcam, USA), mouse anti-Golgin 160 antibody (Santa Cruz, USA), mouse anti-EEA1 antibody (Santa Cruz, USA), mouse anti-GLUT4 antibody (Santa Cruz, USA), mouse anti-TBC1D5 antibody (Santa Cruz, USA), mouse anti-VPS35 antibody (Santa Cruz, USA), mouse anti-VPS29 antibody (Santa Cruz, USA), mouse anti-SNX1 antibody (Santa Cruz, USA), mouse anti-SNX2 antibody (Santa Cruz, USA), mouse anti-SNX5 antibody (Santa Cruz, USA), mouse anti-SNX6 antibody (Santa Cruz, USA), mouse anti-Rab7 antibody (Abcam, USA), HRP-conjugated mouse anti-Actin antibody (Proteintech, USA), mouse anti-α-Tubulin antibody (Proteintech, USA), rabbit anti-ATG5 antibody (Cell Signaling Technology, USA) and rabbit anti-Dynactin p150glued antibody (Cell Signaling Technology, USA).

Cui L., Zhang Q., Huang Y., Yang L., Zhang J., Jiang X., Jia J., Lv Y., Zhang D, & Huang Y. (2021). Impaired Retrograde Transport Due to Lack of TBC1D5 Contributes to the Trafficking Defect of Lysosomal Cathepsins in Ischemic/Hypoxic Cardiomyocytes. Frontiers in Cardiovascular Medicine, 8, 796254.

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Antibody Characterization for Subcellular Studies

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The primary antibodies used in this study included the rabbit anti-LC3B antibody (Sigma), rabbit anti-GFP antibody (Cell Signaling), rabbit anti-p62 antibody (Cell Signaling), rabbit anti-LC3B antibody (Invitrogen), mouse anti-ApoE antibody (Santa Cruz), rabbit anti-ATG5 antibody (Cell Signaling), rabbit anti-HBsAg antibody (Novus Biologicals), mouse anti-CD63 antibody (Santa Cruz), and rabbit anti-Rab11a antibody (Invitrogen). Rabbit antibodies against HBcAg and HBeAg had been described previously (9 (link)). Briefly, the anti-HBcAg was produced by injecting rabbits using core particles expressed in Escherichia coli. The anti-HBeAg was prepared the same way with the exception that the core particles were denatured in SDS-mercaptoethanol prior to injection into rabbits. Secondary antibodies used for immunofluorescence were anti-rabbit and anti-mouse secondary antibodies conjugated with fluorescein isothiocyanate, rhodamine, or Alexa Fluor plus 405 (Invitrogen).

Chu J.Y., Chuang Y.C., Tsai K.N., Pantuso J., Ishida Y., Saito T, & Ou J.H. (2022). Autophagic membranes participate in hepatitis B virus nucleocapsid assembly, precore and core protein trafficking, and viral release. Proceedings of the National Academy of Sciences of the United States of America, 119(30), e2201927119.

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