LC3B (D11) XP® Rabbit mAb (Biotinylated)

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	For Western Blotting dilute at 1:1000

Protocol tips
For Western Blotting dilute at 1:1000

Publication protocol

Autophagy LC3 flux assays
Flux assays were used to quantify autophagy activity wherein accumulation of autophagy substrates, LC3-II or p62, in the presence of inhibitors of lysosomal proteolysis, ammonium chloride (20 mM) and leupeptin (100 μM), reflects activity. Briefly, cells were cultured in the presence or absence of lysosomal proteases for 2 h following which, cells were collected and lysed and subjected to immunoblotting for LC3-II or p62. Autophagy flux was determined by subtracting the densitometric value of inhibitor-untreated LC3-II or p62 from corresponding inhibitor-treated values.

LC3 co-immunoprecipitation
Fresh livers were homogenized in 500 μl of 50 mM Tris/HCl (pH 8.5), 150 mM NaCl, 1% NP40, 5 mM EDTA supplemented with protease/phosphatase inhibitors. Nuclear fractions were obtained as detailed above. Lysates (500 μg of homogenate or 150 μg of nuclear fractions) were incubated overnight in rotation at 4 °C with 100 μl of protein-A sepharose beads (Sigma-Aldrich) crosslinked to LC3 antibody (MBL International, Woburn, MA, USA). For covalent crosslinking, beads were washed (2,500 r.p.m./5 min at 4 °C) with 0.2 M Borate/3 M NaCl buffer (pH 9.0) and crosslinked with 50 mM dimethyl pimelimidate in Borate buffer (30 min) in rotation at RT. Coupling reactions were stopped with 0.2 M ethanolamine (pH 8.0) for 2 h RT followed by incubation with 200 mM glycine (pH 2.5). Crosslinked beads were incubated with samples in rotation overnight at 4 °C. Bound proteins were eluted by boiling (95 °C for 5 min) in 2 × SDS–PAGE sample buffer. Immunoprecipitated (IP) proteins and original lysates (input) were resolved on a SDS–PAGE, and membranes were probed for bRAF, MEK, ERK, P-ERK and LC3. P-ERK1, P-ERK2, P-ERK1/2 or P-MEK1/2 bands obtained by immunoblotting were quantified using ImageJ and normalized to corresponding total ERK1, total ERK2, total ERK1/2 or total MEK1/2, respectively. Full scans of western blots are supplied in Supplementary Fig. S12. The antibody concentrations are supplied in Supplementary Table S1.

Fluorescence microscopy
Cells on coverslips were fixed with a 4% paraformaldehyde solution, blocked and incubated with primary and corresponding secondary antibodies (Alexa Fluor 488 and/or Alexa Fluor 647 conjugated) (Invitrogen). Mounting medium contained DAPI (4',6-diamidino-2-phenylindole) to visualize the nucleus (Invitrogen). Images were acquired on a Leica DMI6000B microscope/ DFC360FX 1.4-megapixel monochrome digital camera (Leica Microsystems, Germany) using × 100 objective/1.4 numerical aperture. Images in each experiment were acquired at same exposure times within the same imaging session. Image slices/stacks of 0.2 μm thickness were captured and deconvolved using the Leica MetaMorph acquisition/analysis software. All images were prepared using Adobe Photoshop and subjected to identical post-acquisition brightness/contrast effects. Representative native and/or inverted images are shown. Quantification was performed in individual frames after deconvolution and thresholding using the ImageJ software (NIH) in a minimum of 20 cells per slide and a minimum of 50 cells from two or more experiments. Particle number was quantified with the ‘analyse particles’ function in threshold single sections with size (pixel2) settings from 0.1 to 10 and circularity from 0 to 1. Cellular fluorescence intensity was expressed as mean integrated density as a function of individual cell size. Nuclear fluorescence intensity was expressed as a function of nuclear size after the identification of the nuclear perimeter by the ImageJ ‘freehand selection tool’. Percentage colocalization was calculated using the JACoP plugin in single Z-stack sections of deconvolved images. Colocalization is shown in merged native images and/or as white pixels using the ‘colocalization finder’ plugin in ImageJ.

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