rabbit anti-lc3 polyclonal antibody mbl pm036  (MBL Life science)

Journal: Nature Communications

Article Title: The T300A Crohn's disease risk polymorphism impairs function of the WD40 domain of ATG16L1

doi: 10.1038/ncomms11821

Figure Lengend Snippet: ( a , b ) Immunoblot analysis of HA-LC3 lipidation induced by TMEM59 overexpression for 36 h in ATG16L1 −/− HCT116 cells restored with full-length HA-ATG16L1 (FL), both ATG16L1 fragments (Nt: 1–299; Ct: 300–607) or irrelevant vector (−). TMEM59-Δ282 is the largest C-terminal deletion that retains the autophagic potential of the molecule. 4M designates a mutated form of TMEM59 where the four residues that are essential for its autophagic activity are mutated to alanine. ( c ) Quantification of GFP-LC3 punctae per transfected cell induced by TMEM59 overexpression for 36 h in the same cell lines as in a and b . Shown are mean values±s.d. ( n =50 cells, *** P <0.001 Student's t -test). ( d , e ) Immunoblot analysis of endogenous LC3 lipidation and p62 expression levels induced in the indicated restored ATG16L1 −/− HCT116 cells by treatment with E64d/Pepstatin (10 μg ml −1 each, 8 h) or bafilomycin (50 nM, 8 h)±rapamycin (2 μg ml −1 , 8 h). ( f ) Quantification of endogenous LC3 punctae per cell induced by the indicated treatments (treatment conditions were as in d and e ; NS, not significant, P >0.05 Student's t -test). All results shown in this figure are representative of at least two repetitions.

Article Snippet: Endogenous LC3 was stained using a rabbit anti-LC3 polyclonal antibody (MBL PM036, 1:600) except for experiments involving infection with S. aureus where a mouse monoclonal anti-LC3 antibody (MBL M152-3 (IgG1 low affinity for protein A) 1:50) was used.

Techniques: Western Blot, Over Expression, Plasmid Preparation, Activity Assay, Transfection, Expressing

Journal: Nature Communications

Article Title: The T300A Crohn's disease risk polymorphism impairs function of the WD40 domain of ATG16L1

doi: 10.1038/ncomms11821

Figure Lengend Snippet: ( a ) Quantification of GFP-LC3 punctae per transfected cell induced by TMEM59 overexpression for 24 h in ATG16L1 −/− HCT116 cells restored with HA-ATG16L1-T300 (T) or HA-ATG16L1-A300 (A). Shown are mean values±s.d. ( n =50 cells, *** P <0.001 Student's t -test). ( b ) Immunoblot analysis of HA-LC3 lipidation induced by TMEM59 overexpression in the same conditions and cell lines as in a . The LC3-II/LC3-I signal intensity ratio is shown at the bottom of the relevant lanes. ( c , d ) Quantification of GFP-LC3 punctae per transfected cell induced by TMEM59 overexpression for 36 h in the same cell lines as in a , and in the absence or presence of zVAD.fmk ( c , 50 μM for the last 8 h of culture), or in ATG16L1 −/− HCT116 cells restored with the indicated ATG16L1 mutants ( d ). Data are displayed as in a (NS, not significant, P >0.05 Student's t -test; *** P <0.001 Student's t -test). ( e ) Representative confocal pictures showing colocalization between TMEM59-GFP and mRFP-LC3 co-expressed for 36 h in the same cell lines as in a . Scale bars represent 5 μm. ( f ) Quantification of the intensity of mRFP-LC3 present in TMEM59-GFP-positive vesicles (mean±s.d., n =100 vesicles present in 8 cells, *** P <0.001 Student's t -test; right panel). Data are presented as a fraction of the mean value obtained in cells expressing ATG16L1-T300. All displayed results are representative of at least two independent repetitions.

Article Snippet: Endogenous LC3 was stained using a rabbit anti-LC3 polyclonal antibody (MBL PM036, 1:600) except for experiments involving infection with S. aureus where a mouse monoclonal anti-LC3 antibody (MBL M152-3 (IgG1 low affinity for protein A) 1:50) was used.

Techniques: Transfection, Over Expression, Western Blot, Expressing

Journal: Nature Communications

Article Title: The T300A Crohn's disease risk polymorphism impairs function of the WD40 domain of ATG16L1

doi: 10.1038/ncomms11821

Figure Lengend Snippet: ( a ) Immunoblot analysis of GSH immunoprecipitates (IP) or total cell lysates (TL) of Atg16l1 −/− MEFs reconstituted with the indicated versions of ATG16L1 (T300 (T) or A300 (A)), retrovirally transduced with TMEM59-GST and subsequently infected (+) with a S. aureus strain (SA; 2 h; multiplicity of infection (m.o.i.)=25) that constitutively expresses GFP, or left uninfected (−). These results are representative of three repetitions. ( b , e ) Quantification of the number of LC3-positive phagosomes containing GFP-positive S. aureus bacteria 2 h after infection (m.o.i.=10) in Atg16L1 −/− MEFs restored with the indicated versions of ATG16L1 (T or A), full-length ATG16L1-T300 (FL), both ATG16L1 fragments that result from caspase-3 processing of the risk allele (Nt: 1–299; Ct: 300–607) or irrelevant vector (−). Values are expressed as a fraction of the scores obtained for cells expressing ATG16L1-T300 (mean±s.d. of triplicates; n =500 cells; *** P <0.001 Student's t -test). ( c , f ) Quantification of the colony-forming units (C.F.U.s) recovered from Atg16L1 −/− MEFs restored with the indicated versions of ATG16L1 (T or A) and infected with S. aureus for 2 h, m.o.i.=1 (mean±s.d., n =6, *** P <0.001, ** P <0.01 Student's t -test). ( d ) Immunoblot analysis of endogenous LC3 activation and p62 expression levels induced by S. aureus infection (2 h, m.o.i.=25) of Atg16l1 −/− MEFs reconstituted with the indicated versions of ATG16L1 (full-length ATG16L1-T300 (FL) or both ATG16L1 fragments that result from caspase-3 processing of the risk allele (Nt: 1–299; Ct: 300–607)), or control vector (−). Results are representative of at least two repetitions.

Article Snippet: Endogenous LC3 was stained using a rabbit anti-LC3 polyclonal antibody (MBL PM036, 1:600) except for experiments involving infection with S. aureus where a mouse monoclonal anti-LC3 antibody (MBL M152-3 (IgG1 low affinity for protein A) 1:50) was used.

Techniques: Western Blot, Transduction, Infection, Bacteria, Plasmid Preparation, Expressing, Activation Assay, Control