Anti-LC3 (Rat) pAb

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	Primary antibody dilution 1:1000

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Primary antibody dilution 1:1000

Publication protocol

Western blot analysis
Total and cytosolic extracts were obtained using a commercial kit (Active Motif, Carlsbad, CA, USA). The following antibodies were used for immunoblotting: rabbit polyclonal anti‐LC3 antibody (1:1000) (Code n.PD014, MBL International Corporation, Woburn, MA, USA), mouse anti‐p62/SQSTM1 antibody (1:250) (Code n.MAB8028, R&D Systems, Minneapolis, MN, USA), and mouse anti‐β‐actin monoclonal antibody (1:5000) (Code n.A5441, Sigma‐Aldrich) as an internal control. Anti‐rabbit (1:2000) (Code n.7074, Chemicon, Temecula, CA, USA) and anti‐mouse (1:10,000) (Code n.103001, BD Pharmingen, Franklin Lakes, NJ, USA) antibodies were used as peroxidase‐conjugated secondary antibodies. Chemiluminescence was detected using luminol solution (ECL Plus, GE Healthcare Amersham, Milan, Italy). Immunoreactive bands were visualized with an exposure time of 15 minutes (Kodak XOMAT). LC3 conversion from LC3 form I (LC3‐I, 18 kDa) to LC3 form II (LC3‐II, 16 kDa), normalized by β‐actin level, was our reported autophagy index.

Immunofluorescence confocal microscopy
Immunofluorescence analysis was carried out to assess the expression of MAP LC3 in bone biopsies and LC3 and Apaf‐1 in the cell cultures. MLO‐Y4 cells, cultured in different conditions in multiwell chamber slides, and bone biopsy samples were treated and observed as previously described.38 Samples were incubated with the following antibodies: rabbit anti‐MAP LC3 (Code n. sc‐134226), mouse anti‐Apaf‐1 (Code n. sc‐135836, Santa Cruz Biotechnology, Inc., Dallas, TX, USA); goat anti‐rabbit Cy3 (Code n. C2306) and sheep anti‐mouse FITC (Code n. F2883, Sigma‐Aldrich). After being washed, samples were counterstained with 1 g/mL DAPI (4',6‐diamidin‐2‐fenilindolo) and then mounted with an anti‐fade medium (0.21M DABCO (1,4‐diazabicyclo[2.2.2]octane) and 90% glycerol in 0.02 M Tris (tris[hydroxymethyl]aminomethane, pH 8.0). The negative control samples were not incubated with the primary antibody. The confocal imaging was performed on a Leica TCS SP2 AOBS confocal laser scanning microscope (Leica S.p.A.). To quantify MAP LC3‐positive cells, 5 to 6 slides were examined at 40× magnification for each condition, in order to count almost 1000 cells as previously described.38 All results are expressed as the mean % ± SD. The mean values of the groups were compared using ANOVA, followed by Bonferroni and Student‐Newman‐Keuls tests

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