Anti mouse 170 6516

Cell pellets were lysed for 30 min in ice-cold whole cell extract buffer (50 mM TrisHCl pH 7.4, 250 mM NaCl, 0.1% Nonidet NP40, 5 mM EDTA, 50 mM NaF and a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Lysates were cleared by centrifuging at 12,000 rpm for 5 min and the protein concentration was determined using a BioRad assay kit (BioRad, Hercules, CA, USA). Cell lysates (50µg) were resolved on 10–12% SDS-PAGE (polyacrylamide gel electrophoresis) gels. Proteins were then transferred to nitrocellulose membranes (Merck Millipore, Burlington, MA, USA). Immunoblotting was carried out with the following antibodies: rabbit anti-PARP #9542 (cell signaling, 1:1000), rabbit anti-γH2AX #9718 (Cell Signaling, Danver, MA, USA, 1:1000), goat anti-ACTIN sc-1615 (Santa Cruz Biotechnology, Dallas, TX, USA, 1:500) and mouse anti-RAN sc-271376 (Santa Cruz Biotechnology, 1:500). The secondary antibodies conjugated with horseradish peroxidase (HRP) anti-rabbit #1706515 and anti-mouse #1706516 were purchased from BIO-RAD Laboratories S.r.l., anti-goat sc-2354 from Santa Cruz Biotechnology. HRP substrate (ECL Western Blotting Detection, Amersham-Life Science, Amersham, UK) was added and the signal was detected with the Odyssey Fc instrument (Li-COR). All the uncut filter and relative densitometric raw data have been included in Figure S7.

Proteins were extracted and visualized as reported in [36 (link)]. Immunoblotting was carried out with the following antibodies: anti-phospho-p44/42 MAPK (ERK 1/2) (Thr202/Tyr204) #9101S, anti-p44/42 MAPK (ERK1/2) #9102S, anti-phospho-S6 Ribosomal Protein (Ser235/236) #221 and anti-S6 Ribosomal protein #2217 purchased from Cell Signaling Technology. Anti-RAN #sc-271376 was purchased from Santa Cruz Biotechnology. The secondary antibodies anti-rabbit #170–6515 and anti-mouse #170–6516 from Bio Rad were used.

RA was purchased from Calbiochem (Millipore Corp). Antibodies against FABP5 (AF1476) and PPARβ/δ (AB10094) were obtained from R&D Systems and Millipore Corp., respectively. Antibodies against RARγ (sc-7387), actin (sc-47778), PCAF (sc-8999), p65 (sc-372), and KLF2 (sc-28675) were purchased from Santa Cruz. Antibodies against PARP (9532) were from Cell Signaling. Anti-mouse (170-6516) and anti-rabbit (170-6515) immunoglobulin horseradish peroxidase-conjugated antibodies were from BioRad. Antibodies against CRABP2 were kindly provided by Cecile Rochette-Egly (IGBMC, Strasbourg, France). Normal mouse (sc-2025) and rabbit (sc-2027) IgG for the Chip control were purchased from Santa Cruz.

HDF cells were seeded at a density of 8 × 103 in 96-well plates and treated with cardoon calli extracts or transforming growth factor-β (TGFβ) (2.5 ng/mL) as positive control. After the treatments, the medium was removed and the cells were washed with PBS 1x, fixed in paraformaldehyde (PFA) 4% for 10 min, and then incubated for 1 h with BSA 3% to saturate non-specific bonds. The cells were then incubated for 2 h with ProCollagen I specific antibody (sc-166572 Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1:500 in PBS 1x, containing 1% BSA. After 3 washes, secondary antibody (anti-mouse 170-6516 Biorad) diluted in PBS containing 1% of BSA was added. Plates were washed 3 times with PBS 1x, one hour later, and the amount of ProColI produced by the cells was measured by a colorimetric reaction, using a 0.35 mg mL⁻¹ solution of o-phenylenediamine (OPD) (Sigma-Aldrich, St. Louis, MO, USA) and 0.012%  of H₂O₂ in citrate buffer 50 mM. The developed color was measured by reading the absorbance at 490 nm using the multiplate reader Victor Nivo (Perkin Elmer, Waltham, MA, USA). The signal for each sample was compared to cellular density determined by crystal violet staining (Sigma-Aldrich, St. Louis, MO, USA).