Anti histone h3

Total proteins from RAGE and/or CHI3L1 expression plasmid transfected cells were extracted using 0.1% NP-40 lysis buffer as previously described [35 (link)]. Nuclear proteins were extracted, incubating cells with hypotonic buffer (20 mM Tris-HCl pH 7,4, 10 mM NaCl, 3 mM MgCl₂) for 15 minutes on ice and centrifuge at 3000 rpm for 10 min at 4°C. The pellet was then lysed with 1% NP-40 lysis buffer for 30 minutes on ice and supernatants were collected by centrifuging at 13,000 rpm for 30 minutes [35 (link)]. SDS-PAGE and Western blot were performed as previously described using anti-phospho/total STAT3 (Cell Signaling, Danvers, MA), anti-β-catenin (Abcam), anti-Histone H3 (Cell Signaling), and anti-p65 (Santa Cruz, Dallas, Tx) primary antibodies [36 (link)].

3-(4,5-dimethylthiazol-diphenyltetrazolium bromide (MTT), was purchased from Sigma Aldrich (Milan, Italy). The following rabbit polyclonal antibodies (Abs) were used: Anti-histone H3 (1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-COX IV (1:250; Cell Signaling Technology) and anti-caveolin-1 (1:400; Cell Signaling Technology). The following mouse monoclonal Abs were used: anti-TRPML1 (clone F-10, 1:300 for Western blot, 1:50 for FACS analysis; Santa Cruz Biotechnology, Dallas, TX, USA), anti-TRPML2 (1:300 for Western blot; Santa Cruz Biotechnology), anti-LAMP1 (1:300; Santa Cruz Biotechnology), anti-calreticulin (1:1000; BD Biosciences, Milan, Italy) and anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH, 14C10, 1:1000; Cell Signaling Technology). anti-TRPML2 from Sigma Aldrich was used diluted 1:50 for FACS analysis.
The following secondary antibodies were used: horseradish peroxidase (HRP)-conjugated anti-mouse IgG and HRP-conjugated anti-rabbit IgG (1:2000; Cell Signaling Technology), FITC-conjugated anti-rabbit Ab (BD Biosciences), PE-conjugated anti-mouse Ab (BD Biosciences, Milan, Italy), Alexa Fluor-594-conjugated anti-mouse Ab (1:100; Invitrogen, San Diego, CA, USA), Alexa Fluor-488-conjugated anti-mouse Ab (1:100; Invitrogen) and Alexa Fluor-488-conjugated anti-rabbit Ab (1:100; Invitrogen).

Cells were collected in RIPA buffer (10 mM Tris-HCl (pH 8.0), 1% (w/v) NP40, 0.1% (w/v) sodium deoxycholate (Wako), 0.1% (w/v) SDS (Wako), 0.15 M NaCl (Wako), 1 mM EDTA, 10 mM NaF (Wako), 1.5 mM Na₃VO₄ (Wako), and cOmplete™ Protease Inhibitor Cocktail (Roche)). Protein concentrations were titered using the BCA protein assay kit (Thermo Fisher Scientific). Collected protein lysate was mixed with SDS-PAGE loading buffer (0.15 M Tris-HCl, 6% (w/v) SDS, 0.003% (w/v) bromophenol blue (Wako), 30% (w/v) glycerol (Wako), and 15% (w/v) β-mercaptoethanol (Wako)) and then boiled at 95 °C for 5 min, followed by SDS-PAGE and immunoblotting. Antibodies used for immunoblotting were as follows: anti-milk (rabbit, 1:1000; Nordic-MUbio, Susteren, the Netherlands), anti-PyMT (rat, 1:500; Santa Cruz), anti-mCherry (rabbit, 1:500; Abcam), anti-histone H3 (rabbit, 1:2500; Cell Signaling Technology, MA, USA), and anti-α-tubulin (mouse, 1:5000; Calbiochem).