polyclonal antibodies against atg5  (Novus Biologicals)

Journal: Veterinary research

Article Title: Incomplete autophagy promotes the proliferation of Mycoplasma hyopneumoniae through the JNK and Akt pathways in porcine alveolar macrophages.

doi: 10.1186/s13567-022-01074-5

Figure Lengend Snippet: Figure 1 M. hyopneumoniae infection increases the formation of autophagosome-like vesicles and the expression of autophagy-related proteins in porcine alveolar macrophages. A Autophagosome-like vesicles (arrows) were observed in M. hyopneumoniae-negative (a) and M. hyopneumoniae-positive (b) PAMs by TEM. Ten M. hyopneumoniae-negative and 10 M. hyopneumoniae-positive PAMs were assessed. B Autophagosome-like vesicles (arrows) were observed in mock-infected (a) or AH-infected (b) 3D4/21 cells by TEM. Ten uninfected and 10 AH-infected 3D4/21 cells were assessed. C Results of Western blotting showing the expression of autophagy-related proteins (LC3-II, Atg5, Atg12-Atg5, and Beclin 1) and Mhp366 in 30 M. hyopneumoniae-positive and 10 M. hyopneumoniae-negative PAMs collected from pigs. D Results of Western blotting showing the expression of autophagy-related proteins (LC3-II, Atg5, Atg12-Atg5, and Beclin 1) and P97 protein in 3D4/21 cells after mock infection or AH infection. Mean values from 3 independent experiments are presented. Between-group differences were assessed using a t test; *p ≤ 0.05, **p ≤ 0.01.

Article Snippet: Polyclonal antibodies against ATG5 (NBP2-24389), ATG5 (NB11053818), Beclin 1 (NB110-87318), and p62 (NBP1-48320) were purchased from Novus Biologicals (Shanghai, China).

Techniques: Infection, Expressing, Western Blot

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: Pioglitazone Reduces Hepatic Alpha-1 Antitrypsin Accumulation Through Autophagy and AMPK Activation in Alpha-1 Antitrypsin Deficient Mice

doi: 10.1152/ajpgi.00272.2025

Figure Lengend Snippet: (A) Western blot analysis of autophagy-related proteins in HuhZ cells treated with pioglitazone (10 μM, 48 h), with or without Bafilomycin A1 (BafA1, 100 μM, 6 h). Protein levels of ATG5, p62, and LC3B were assessed. GAPDH serves as a loading control. (B) Quantification of p62 and LC3B-II band intensities from (A), normalized to GAPDH. Bars represent mean ± SD from three independent experiments. (C) Immunofluorescence staining of LC3B (green) in HuhZ cells treated with pioglitazone ± BafA1. Nuclei were counterstained with DAPI (blue). Insets show enlarged views of LC3B-positive puncta. (D) Western blot analysis of AMPK-mTOR signaling components in control and pioglitazone-treated HuhZ cells. Phosphorylation of AMPK, ULK1, and mTOR was assessed. (E) Quantification of p-AMPK, p-ULK1, and p-mTOR band intensities from (D), normalized to GAPDH. Bars show mean ± SD. (F) Immunofluorescence staining of p-AMPK (green) and actin (red) in control and pioglitazone-treated HuhZ cells. Nuclei were counterstained with DAPI (blue). Merged images show increased p-AMPK activation upon pioglitazone treatment.

Article Snippet: The blots were blocked in 5% non-fat dried milk and incubated overnight at 4°C with rabbit polyclonal antibodies against ATG5, AMPK, p-AMPK, ULK, p-ULK, mTOR, p-mTOR, PTEN, and p-PTEN (Cell Signaling Technology, Danvers, MA, USA); LC3 (Proteintech, Rosemont, IL); and AAT (Dako, Carpenteria, CA).

Techniques: Western Blot, Control, Immunofluorescence, Staining, Phospho-proteomics, Activation Assay

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: Pioglitazone Reduces Hepatic Alpha-1 Antitrypsin Accumulation Through Autophagy and AMPK Activation in Alpha-1 Antitrypsin Deficient Mice

doi: 10.1152/ajpgi.00272.2025

Figure Lengend Snippet: (A) Western blot analysis of liver lysates from control- and pioglitazone-treated Pi*Z mice (30 mg/kg/day, 12 weeks) showing levels of total AAT, ATG5, LC3B-I/II, and p62. GAPDH is shown as a loading control. (B) Quantification of Western blot band intensities from (A), normalized to GAPDH. Data represents SD from 4 mice per group. Statistical significance assessed by unpaired t-test. (C) Immunohistochemistry for LC3B in liver sections from control and pioglitazone-treated mice. The right panel shows higher magnification with arrows indicating LC3B-positive vacuoles. (D) Immunohistochemistry for p62 in liver sections from control and pioglitazone-treated mice. p62-positive aggregates are reduced in pioglitazone-treated livers. Scale bars: 100 μm (all panels).

Article Snippet: The blots were blocked in 5% non-fat dried milk and incubated overnight at 4°C with rabbit polyclonal antibodies against ATG5, AMPK, p-AMPK, ULK, p-ULK, mTOR, p-mTOR, PTEN, and p-PTEN (Cell Signaling Technology, Danvers, MA, USA); LC3 (Proteintech, Rosemont, IL); and AAT (Dako, Carpenteria, CA).

Techniques: Western Blot, Control, Immunohistochemistry

Journal: International Dental Journal

Article Title: Yunnan Baiyao Inhibits Periodontitis by Suppressing the Autophagic Flux

doi: 10.1016/j.identj.2023.09.005

Figure Lengend Snippet: Role of autophagy in the Yunnan Baiyao (YNBY)–mediated inhibition of osteoclast differentiation. A, B, GFP-RFP-LC3 fluorescence staining revealed that RANKL increased the formation of autophagolysosomes, whilst excluding the effect of solvent dimethylsulfoxide on autophagic flux. C–F, YNBY prevented osteoclast differentiation and F-actin ring formation by inhibiting autophagy. * P < .05. ** P < .01.

Article Snippet: Rabbit polyclonal antibodies against LC3 (#14600-1-AP), ATG5 (#10181-2-AP), Beclin1 (#11306-1-AP), P62 (#18420-1-AP), CTSK (#11239-1-AP), and NFATc-1 (#66963-1-Ig) were acquired from Proteintech Group (Wuhan, China).

Techniques: Inhibition, Fluorescence, Staining, Solvent

Journal: International Dental Journal

Article Title: Yunnan Baiyao Inhibits Periodontitis by Suppressing the Autophagic Flux

doi: 10.1016/j.identj.2023.09.005

Figure Lengend Snippet: Effect of Yunnan Baiyao (YNBY) on the autophagic flux during osteoclast differentiation. A, B, Bone marrow–derived macrophage (BMM) cells were treated with 20 µg/mL YNBY, 1 mmol/L 3-methyladenine (3-MA), or 100 nmol/L rapamycin (RAP) for 6 hours, and then cells were stained with LysoTracker Red and monitored using confocal immunofluorescence microscopy (scale bar = 100 μm). C, Transmission electron microscopy images of BMMs; left panels (magnification, 10,000x, scale bar = 2 μm) and right panels (magnification, 25,000×, scale bar = 500 nm). Autophagosomes are indicated by yellow arrows. D, E, After infecting BMMs with GFP-RFP-LC3 tandem fluorescent protein adenovirus, the cells were incubated with osteoclast medium supplemented with 20 µg/mL YNBY, 1 mmol/L 3-MA, or 100 nmol/L RAP for 6 hours. Then, fluorescence was observed with a confocal microscope, and quantitative analysis was performed. Scale bar = 100 μm. The data are presented as mean ± SDs of 3 independent experiments (* P < .05, ** P < .01). All the experiments were carried out independently at least 3 times (* P < .05, ** P < .01; bar = 100 μm).

Article Snippet: Rabbit polyclonal antibodies against LC3 (#14600-1-AP), ATG5 (#10181-2-AP), Beclin1 (#11306-1-AP), P62 (#18420-1-AP), CTSK (#11239-1-AP), and NFATc-1 (#66963-1-Ig) were acquired from Proteintech Group (Wuhan, China).

Techniques: Derivative Assay, Staining, Immunofluorescence, Microscopy, Transmission Assay, Electron Microscopy, Incubation, Fluorescence

Journal: International Dental Journal

Article Title: Yunnan Baiyao Inhibits Periodontitis by Suppressing the Autophagic Flux

doi: 10.1016/j.identj.2023.09.005

Figure Lengend Snippet: Yunnan Baiyao (YNBY) inhibited bone resorption and the expression of osteoclast-related proteins and autophagy-related proteins. A, B, Bone marrow–derived macrophages (BMMs) were seeded on bovine bone slices and allowed to adhere to the surface. BMMs were pretreated with 1 mmol/L 3-methyladenine (3-MA) or 100 nmol/L rapamycin (RAP) for 1 hour and then cultured with osteoclast medium for 5 days. Representative scanning electron microscopy images of bone resorption pits are shown. C, D, BMMs were pretreated with 1 mmol/L 3-MA or 100 nmol/L RAP for 1 hour and then cultured with RANKL for 6 hours. The total proteins were extracted to measure the levels of P62 and LC3. The total proteins were extracted on the 5th day, and western blotting was performed to examine the expression of the osteoclast-related proteins NFATc-1 and CTSK. E, Schematic model of the hypothesised mechanism by which YNBY inhibits osteoclast differentiation. Densitometric analysis of an immunoblot from 3 independent experiments. * P < .05. ** P < .01.

Article Snippet: Rabbit polyclonal antibodies against LC3 (#14600-1-AP), ATG5 (#10181-2-AP), Beclin1 (#11306-1-AP), P62 (#18420-1-AP), CTSK (#11239-1-AP), and NFATc-1 (#66963-1-Ig) were acquired from Proteintech Group (Wuhan, China).

Techniques: Expressing, Derivative Assay, Cell Culture, Electron Microscopy, Western Blot