rabbit polyclonal anti human lc3 b antibody  (Bio-Techne corporation)

Journal: Biomedicines

Article Title: mTOR Inhibitors Modulate the Biological Nature of TGF-β2-Treated or -Untreated Human Trabecular Meshwork Cells in Different Manners

doi: 10.3390/biomedicines12112604

Figure Lengend Snippet: Western blotting of 2D HTM cells. Two-dimensional HTM cells treated or not treated with 5 ng/mL TGF-β2 and/or 100 nM of an mTOR inhibitor, Rapa or Torin1, for 6 days were subjected to Western blotting using antibodies (1:1000 dilutions) against LC3 (LC3-I; 16kDa, LC3-II; 13 kDa) and α-tubulin (55 kDa). The experiments were performed in triplicate using fresh preparations.

Article Snippet: After blocking had been performed with a TBS-T buffer containing 5% nonfat dry milk or 5% BSA, the blots were incubated with 1:1000 dilutions of anti-human LC3 rabbit polyclonal antibody (Cell Signaling Technology, Beverly, MA, USA) or 1:1000 dilutions of anti-human α-tubulin mouse monoclonal antibody (Sigma Aldrich, St. Louis, MO, USA) at 37 °C for 2 h. Then, they were incubated with an HRP-conjugated secondary antibody, goat anti-rabbit antibody, or goat anti-mouse antibody (Biorad Laboratories Inc., Hercules, CA, USA) at 37 °C for 1 h after washing three times with TBS-T buffer for 10 min each time.

Techniques: Western Blot

Journal: Oncology reports

Article Title: Inhibition of PIKfyve using YM201636 suppresses the growth of liver cancer via the induction of autophagy.

doi: 10.3892/or.2018.6928

Figure Lengend Snippet: Figure 3. YM201636 significantly increased the levels of LC3II. (A) HepG2 and (B) Huh‑7 cells. β‑actin was used as an internal control. Naïve cells were untreated. ***P<0.001 vs. DMSO group, ###P<0.001 vs. naive group, one‑way analysis of variance followed by a Dunnett's test, n=5/group. DMSO, dimethyl sulfoxide; LC3, microtubule associated protein 1 light chain 3.

Article Snippet: China E-mail: xiesq@vip.henu.edu.cn Key words: YM201636, autophagy, epidermal growth factor receptor, hepatoma Santa Cruz Biotechnology, Inc. Polyclonal rabbit anti-human microtubule associated protein 1 light chain 3 (LC3) A/B antibody (cat. no. 14600-1-AP; 1:1,000) was purchased from ProteinTech Group, Inc. (Chicago, IL, USA).

Techniques: Control

Journal: Oncology reports

Article Title: Inhibition of PIKfyve using YM201636 suppresses the growth of liver cancer via the induction of autophagy.

doi: 10.3892/or.2018.6928

Figure Lengend Snippet: Figure 8. EGFR overexpression significantly increases the levels of LC3II. Western blot analysis of LC3 in (A) HepG2 and (B) Huh‑7 cells. β‑actin was used as an internal control. Naïve cells were untreated. ***P<0.001 vs. vector group, ###P<0.001 vs. naive group, one‑way analysis of variance, n=5/group. EGFR, epidermal growth factor receptor; LC3, microtubule associated protein 1 light chain 3.

Article Snippet: China E-mail: xiesq@vip.henu.edu.cn Key words: YM201636, autophagy, epidermal growth factor receptor, hepatoma Santa Cruz Biotechnology, Inc. Polyclonal rabbit anti-human microtubule associated protein 1 light chain 3 (LC3) A/B antibody (cat. no. 14600-1-AP; 1:1,000) was purchased from ProteinTech Group, Inc. (Chicago, IL, USA).

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

Journal: Nanomaterials

Article Title: The Effects of Silica Nanoparticles on Apoptosis and Autophagy of Glioblastoma Cell Lines

doi: 10.3390/nano7080230

Figure Lengend Snippet: Western blot analysis ( A ) and densitometric analysis ( B ) of LC3-I and LC3-II ratio in LBC3 cells incubated in medium with 50 and 100 µg/mL of 5–15 nm SiNPs for 24 and 48 h. Samples containing 20 μg of protein were submitted to electrophoresis and immunoblotting. A representative Western blot is presented on panel A and densitometric analysis—on panel B. Relative quantification of Atg5 gene expression in LBC3 cells ( C ). Results are shown as a relative fold change in mRNA expression in comparison to untreated controls, where expression level was set as 1. The effect of SiNPs on formation of AVOs in the LBC3 cell lines ( D , E ) evaluated by fluorescence microscope assay. The volume of the cellular acidic compartment was visualized by acridine orange staining. The cells were incubated in DMEM with 50 or 100 μg/mL of 5–15 nm SiNPs for 24 or 48 h and the cells were photographed under a fluorescence microscope at 200-fold magnification, ( scale bar 50 μm) ( D ), or counted was the percentage of AVOs-positive cells ( E ). Representative images of AVOs positive cells (red arrows), from one of three independent experiments are shown. Significant alterations are expressed relative to controls and marked with asterisks. Mean values from three independent experiments ± SD are presented; * p < 0.05 or ** p < 0.001.

Article Snippet: Polyclonal (rabbit) anti-human LC3 antibody and alkaline phosphatase-labeled anti-rabbit immunoglobulin G were provided by Cell Signaling Technology (Boston, MA, USA).

Techniques: Western Blot, Incubation, Electrophoresis, Quantitative Proteomics, Gene Expression, Expressing, Comparison, Fluorescence, Microscopy, Staining

Journal: Frontiers in Neuroscience

Article Title: ASPP2 Plays a Dual Role in gp120-Induced Autophagy and Apoptosis of Neuroblastoma Cells

doi: 10.3389/fnins.2017.00150

Figure Lengend Snippet: HIV-1 gp120 can induce autophagy and apoptosis in SH-SY5Y neuroblastoma cells. (A) Bands of autophagic proteins of LC3-I/LC3-II, Beclin1, and beta-actin treated by different concentrations of HIV-1 gp120. (B) Data of the LC3-II/actin. (C) Beclin1 were normalized with beta-actin and analyzed with ImageJ software (National Institutes of Health). (D) Autophagosomes formation in SH-SY5Y cells treated by HIV-1 gp120 with different concentrations (100× objective lens) and cell viability of SH-SY5Y cells treated by HIV-1 gp120 with different concentrations (cells with green color were alive and cells with red color were dead or apoptosis; 40× objective lens). (E) The mean levels of GFP-LC3 positive puncta with different treatments. (F) The mean levels of cell mortality with different treatments by Calcein AM/PI kit. (G) The mean levels of apoptosis cells with different treatments by Annexin V/7-aad kit. All data were shown as the mean ± S.E.M. of three independent experiments. * P < 0.05; ** P < 0.01.

Article Snippet: The membrane was blocked with 5% non-fat milk, probed with rabbit polyclonal antibodies against human LC3 or cleaved LC3 (LC3-II) or Beclin 1 (Cell Signaling Technology, USA), mouse monoclonal antibody against human β-actin (Cell Signaling Technology, USA), and ASPP2 (sigma, Germany).

Techniques: Software

Journal: Frontiers in Neuroscience

Article Title: ASPP2 Plays a Dual Role in gp120-Induced Autophagy and Apoptosis of Neuroblastoma Cells

doi: 10.3389/fnins.2017.00150

Figure Lengend Snippet: Overexpression of ASPP2 inhibits autophagy in SH-SY5Y neuroblastoma cells when treated by 50 ng/mL gp120 but promotes autophagy when treated by 200 mg/ml gp120. (A) Bands of ASPP2, Beclin1, LC3-I/LC3-II of SH-SY5Y cells which were infected with ASPP2-rAd and the control rAd for 36 h then treated by 50 and 200 ng/mL gp120, respectively. (B) Data of the LC3-II/actin. (C) Relative levels of Beclin1 normalized with beta-actin. (D) The detection of cells with GFP-LC3 positive puncta when treated by 50 and 200 ng/mL gp120, respectively. Representative images are shown (with 100× objective lens). (E) The mean levels of GFP-LC3 positive puncta cells treated with different concentrations of gp120. A total of 100 cells were counted in each group. All data were shown as the mean ± S.E.M. of three independent experiments, * p < 0.05, ** p < 0.01.

Article Snippet: The membrane was blocked with 5% non-fat milk, probed with rabbit polyclonal antibodies against human LC3 or cleaved LC3 (LC3-II) or Beclin 1 (Cell Signaling Technology, USA), mouse monoclonal antibody against human β-actin (Cell Signaling Technology, USA), and ASPP2 (sigma, Germany).

Techniques: Over Expression, Infection, Control

Journal: Frontiers in Neuroscience

Article Title: ASPP2 Plays a Dual Role in gp120-Induced Autophagy and Apoptosis of Neuroblastoma Cells

doi: 10.3389/fnins.2017.00150

Figure Lengend Snippet: The silence of ASPP2 promoted autophagy and apoptosis in SH-SY5Y cells in the presence of low dose gp120 and decreased autophagy and apoptosis in the presence of high dose gp120. (A) Bands of ASPP2, Beclin1, and LC3-I/LC3-II of SH-SY5Y cells which were transfected by ASPP2 siRNA and control siRNA then treated by 50 ng/mL gp120. (B) Relative levels of Beclin1. (C) Data of the LC3-II/actin in different groups. (D) Detection of cells with GFP-LC3 positive puncta of autophagy in different group. (E) The mean levels of cells with autophagy in different groups, a total of 100 cells were counted in each group. (F) Bands of ASPP2, Beclin1, and LC3-I/LC3-II of SH-SY5Y cells which were transfected by ASPP2 siRNA and control siRNA then treated by 200 gp120. (G) Relative levels Beclin1 in different groups when treated by 200 gp120. (H) Data of LC3-II/actin in different groups. (I) Detection of SH-SY5Y cells with GFP-LC3 positive puncta of autophagy in different groups when treated by 200 ng/mL gp120. (J) The mean levels of autophagy cells in different groups when treated by 200 ng/mL gp120. A total of 100 cells were counted in each group and data was shown as the mean ± S.E.M of three independent experiments. * P < 0.05; ** P < 0.01.

Article Snippet: The membrane was blocked with 5% non-fat milk, probed with rabbit polyclonal antibodies against human LC3 or cleaved LC3 (LC3-II) or Beclin 1 (Cell Signaling Technology, USA), mouse monoclonal antibody against human β-actin (Cell Signaling Technology, USA), and ASPP2 (sigma, Germany).

Techniques: Transfection, Control