LC3B Antibody Kit for Autophagy

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Antibody detects two bands corresponding to LC3b-I and LC3b-II.	1:1500 dilution of primary antibody

Upstream tips
Antibody detects two bands corresponding to LC3b-I and LC3b-II.
Protocol tips
1:1500 dilution of primary antibody

Publication protocol

Total proteins were isolated from scraped intestinal mucosa of 180 to 210 day-old mice and control littermates with RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP40, 0.5% Triton X-100, 1 mM EDTA, 0.2% SDS, 0.5% Na-deoxycholate) containing protease and phosphatase inhibitors . Thirty µg of protein extract were analyzed by 10% BisTris NuPAGE (Invitrogen) and transferred onto a PVDF blotting membrane (Roche Diagnostics, QC), after which Western blotting was performed as described , . The following affinity-purified antibodies were used: claudin-1 rabbit polyclonal antibodies (1∶500), claudin-2 rabbit polyclonal antibodies (1∶1000), occludin rabbit polyclonal antibodies (1∶500), JAM-A rabbit polyclonal antibodies (1∶500) from Zymed Laboratories (Invitrogen); LC3b-I and LC3b-II rabbit polyclonal antibodies (1∶1500), E-cadherin rabbit polyclonal antibodies (1∶1000), IRE1α rabbit polyclonal antibodies (1∶500) from Cell Signaling; p62 mouse monoclonal antibodies (1∶1000) from Abcam, and actin (1∶10 000), defensin 4 goat polyclonal antibodies (1∶250), c-Myc rabbit polyclonal antibodies (1∶200) and Cyclin D1/D2 rabbit polyclonal antibodies (1∶500) from Santa Cruz. For densitometry analyses, exposed films of Western blots were scanned and images were analyzed using ImageJ (Rasband, WS, ImageJ, US National Institutes of Health, Bethesda, Maryland, USA).

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