the anti‐lc3 antibody (cat. al221)  (Beyotime)

Journal: Journal of Cellular and Molecular Medicine

Article Title: TLR3 contributes to persistent autophagy and heart failure in mice after myocardial infarction

doi: 10.1111/jcmm.13328

Figure Lengend Snippet: A TLR3 agonist polyinosinic‐polycytidylic acid (poly(I:C)) induced autophagy in cultured cardiomyocytes through a TRIF‐dependent pathway. ( A ) Poly(I:C) increased autophagy markers in cultured H9c2 rat ventricular cells. ( B ) Poly(I:C) stimulated autophagosome formation but did not affect autophagic flux. Primary cultured neonatal rat ventricular myocytes (NRVMs) were transfected with a tandem mRFP‐GFP‐LC3 adenovirus for 24 hrs, followed by treatment with poly(I:C) (100 μg/ml, 4 hrs). Autophagosomes and autolysosomes were, respectively, visualized as yellow‐ and red‐only punctas under a confocal microscope. ( C ) An autophagic flux inhibitor chloroquine (CQ) induced accumulations of LC3‐II and p62/SQSTM1 proteins in H9c2 myocytes receiving poly(I:C) (100 μg/ml, 4 hrs). CQ was applied at 10 μM, immediately prior to poly(I:C). ( D ) Effects of indicated siRNA on poly(I:C)‐induced changes in autophagy markers in NRVMs. All the siRNAs were transfected at 50 nM for 48 hrs, and poly(I:C) was added at 100 μg/ml for 4 hrs before cell harvest. Negative control (NC) siRNA served as control. RNAiMAX transfection reagent was used in all the siRNA experiments. The upper panel shows the knockdown effects of siRNAs, and the lower panel shows representative Western blot images (presented from four independent experiments) and densitometry quantitative data (normalized into ‘fold of vehicle group’). All quantitative data are expressed as means ± S.D. a P < 0.05, A P < 0.01 versus vehicle; b P < 0.05, B P < 0.01 versus poly(I:C).

Article Snippet: The primary antibodies against TLR3 (Cat. NB100‐56571), MyD88 (Cat. NBP1‐19785) and Trif (Cat. NB120‐13810) were purchased from Novus Biologicals, LLC, Littleton, CO, USA; the anti‐LC3 antibody (Cat. AL221) was from Beyotime Institute of Biotechnology, Jiangsu, China; the anti‐beclin‐1 antibody (Cat. 11306‐1‐AP) was from Proteintech Group, Inc., Rosemont, IL, USA; and the anti‐p62 antibody (Cat. 5114) was from Cell Signalling Technology, Inc., Danvers, MA, USA.

Techniques: Cell Culture, Transfection, Microscopy, Negative Control, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: TLR3 contributes to persistent autophagy and heart failure in mice after myocardial infarction

doi: 10.1111/jcmm.13328

Figure Lengend Snippet: Autophagy induction abolished the protection of TLR3‐KO against MI. An autophagy inducer rapamycin (Rapa, 2 mg/kg/day) or an autophagic flux inhibitor chloroquine (CQ, 50 mg/kg/day) was daily intraperitoneally injected for 2 weeks, starting from day 1 after surgery. Normal saline (NS) was injected as control. Measurements were taken at 2 weeks. ( A ) Representative Western blot images and quantitative data of LC3 and p62 proteins in infarct tissue. Rapamycin increased LC3‐II in all the groups, suggesting successful induction of autophagy. CQ (blue bars) induced similar accumulations of LC3‐II and p62 in WT and TLR3‐KO myocardium, suggesting that autophagy flux was comparable between the two groups. ( B ) Representative coronal‐sectional images of Masson's trichrome staining and infarct size of post‐infarct hearts. ( C ) Fractional shortening (FS, %) of the left ventricle. All data are means ± S.D. n = 4–6 mice/group. A P < 0.01 versus respective ‘sham+NS’; b P < 0.05, B P < 0.01 versus respective ‘MI+NS’.

Article Snippet: The primary antibodies against TLR3 (Cat. NB100‐56571), MyD88 (Cat. NBP1‐19785) and Trif (Cat. NB120‐13810) were purchased from Novus Biologicals, LLC, Littleton, CO, USA; the anti‐LC3 antibody (Cat. AL221) was from Beyotime Institute of Biotechnology, Jiangsu, China; the anti‐beclin‐1 antibody (Cat. 11306‐1‐AP) was from Proteintech Group, Inc., Rosemont, IL, USA; and the anti‐p62 antibody (Cat. 5114) was from Cell Signalling Technology, Inc., Danvers, MA, USA.

Techniques: Injection, Western Blot, Staining